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Plant Physiol. Sep 2010; 154(1): 245–261.
Published online Jul 19, 2010. doi:  10.1104/pp.110.162339
PMCID: PMC2938166

CaMsrB2, Pepper Methionine Sulfoxide Reductase B2, Is a Novel Defense Regulator against Oxidative Stress and Pathogen Attack1,[C][W]

Abstract

Reactive oxygen species (ROS) are inevitably generated in aerobic organisms as by-products of normal metabolism or as the result of defense and development. ROS readily oxidize methionine (Met) residues in proteins/peptides to form Met-R-sulfoxide or Met-S-sulfoxide, causing inactivation or malfunction of the proteins. A pepper (Capsicum annuum) methionine sulfoxide reductase B2 gene (CaMsrB2) was isolated, and its roles in plant defense were studied. CaMsrB2 was down-regulated upon inoculation with either incompatible or compatible pathogens. The down-regulation, however, was restored to the original expression levels only in a compatible interaction. Gain-of-function studies using tomato (Solanum lycopersicum) plants transformed with CaMsrB2 resulted in enhanced resistance to Phytophthora capsici and Phytophthora infestans. Inversely, loss-of-function studies of CaMsrB2 using virus-induced gene silencing in pepper plants (cv Early Calwonder-30R) resulted in accelerated cell death from an incompatible bacterial pathogen, Xanthomonas axonopodis pv vesicatoria (Xav) race 1, and enhanced susceptibility to a compatible bacterial pathogen, virulent X. axonopodis pv vesicatoria race 3. Measurement of ROS levels in CaMsrB2-silenced pepper plants revealed that suppression of CaMsrB2 increased the production of ROS, which in turn resulted in the acceleration of cell death via accumulation of ROS. In contrast, the CaMsrB2-transgenic tomato plants showed reduced production of hydrogen peroxide. Taken together, our results suggest that the plant MsrBs have novel functions in active defense against pathogens via the regulation of cell redox status.

Aerobic organisms cannot exist without oxygen, yet paradoxically, oxygen is inherently dangerous to their existence due to the possibility of the conversion of oxygen to reactive oxygen species (ROS) in biological pathways (Davies, 1995). It is due to their highly reactive nature that hydrogen peroxide (H2O2), superoxide anion (O2·), and hydroxyl radical are termed ROS (Halliwell and Gutteridge, 1999). Excessive amounts of ROS in living organisms can cause severe injury or even death by damaging lipids, proteins, carbohydrates, and nucleic acids (Monk et al., 1989; Wu et al., 2005).

The attack of ROS on protein induces the oxidation of Met to Met sulfoxide (MetSO; Gao et al., 1998). Met is the most sensitive amino acid to oxidation, and the oxidation of Met to MetSO can lead to changes in hydrophobicity, alterations in protein conformation, and even loss of biological activity of the proteins (Stadtman et al., 2003; Ezraty et al., 2005; Tarrago et al., 2009). The nonenzymatic oxidation of surface-exposed Met residues of proteins by ROS produces mixtures of two diastereoisomers of MetSO, referred to as Met-S-SO and Met-R-SO, due to the asymmetric position of the sulfur atom in the lateral chain (Stadtman et al., 2003). Because of the toxicity arising from the oxidation of Met, living organisms have developed Met sulfoxide reductases (Msr) to repair the oxidized Met. Msr reverts MetSO to Met (Ezraty et al., 2005; Stadtman et al., 2005; Tarrago et al., 2009) and could participate in the scavenging of ROS and reactive nitrogen intermediates in cells by reversible oxidation/reduction of Met in proteins (Levine et al., 1999). There are two distinct isozymes of Msr, Met sulfoxide reductase A (MsrA) and Met sulfoxide reductase B (MsrB), specific for the conversion of the S and R diastereomer forms of MetSO, respectively (Stadtman et al., 2005). MsrA and MsrB do not share homology either at the level of the primary sequence or the tertiary structure (Lowther et al., 2002). Orthologs of MsrA and MsrB genes are present in most living organisms, and there is great diversity in the organization and copy number of the Msr genes in different organisms (Ezraty et al., 2005).

In plants, the enzymatic activity of Msr was first reported in 1966 (Doney and Thompson, 1966), whereas the first MsrA and MsrB genes in plants were isolated from Brassica napus in 1996 (Sadanandom et al., 2000) and from Arabidopsis (Arabidopsis thaliana) in 2002 (Rodrigo et al., 2002), respectively. Studies on the MsrA and MsrB genes in Arabidopsis revealed the complexity of these multigene families in various subcellular locations (Sadanandom et al., 2000; Vieira Dos Santos et al., 2005). MsrA in Arabidopsis comprises a multigene family with at least five MsrA genes, which are translocated at the cytosol (three members), chloroplasts (one member), and the secretory pathway (one member; Sadanandom et al., 2000). The cytosolic form is present in all tissues, whereas the chloroplastic form is limited mostly to green tissues (Sadanandom et al., 2000). An Arabidopsis MsrA mutant, lacking a cytosolic MsrA gene (Msr2), showed reduced growth and slower development under short-day conditions but not under long-day conditions (Bechtold et al., 2004). Treatment with methyl viologen (MV) as a producer of O2· and high levels of light led to an increase in the expression of plastidic MsrA in Arabidopsis (Romero et al., 2004). Transgenic Arabidopsis plants overexpressing the plastidic MsrA showed increased tolerance to oxidative stress and decreased sulfoxide content (Romero et al., 2004). The first identified MsrA substrate in plants was Hsp21, a chloroplast-localized small heat shock protein, which can become sulfoxidized in the conserved N-terminal region that is highly rich in Met residues and then reduced by the plastidic MsrA in order to maintain the function of Hsp21 as a chaperone (Gustavsson et al., 2002).

The first plant MsrB gene was identified by in silico analysis of the Arabidopsis genome in a search for selenoproteinX-like proteins, which show significant homology to the human selenoproteinX proteins (Rodrigo et al., 2002). The selenoproteinX-like proteins have MsrB activities in Staphylococcus aureus (Singh et al., 2001; Moskovitz et al., 2002) and in mouse (Moskovitz et al., 2002). Nine open reading frames of MsrB genes are identified in Arabidopsis, with the gene products located in the cytoplasm (six members), chloroplasts (two members), and the secretory pathway (one member; Vieira Dos Santos et al., 2005). The most ubiquitously expressed MsrB shows enhanced expression in response to plant dehydration and oxidative stress, and MsrB knockout mutants of Arabidopsis accumulate higher levels of oxidized Met upon exposure to oxidative stress, suggesting that MsrB is involved in the response to oxidative stress (Rodrigo et al., 2002; Vieira Dos Santos et al., 2005; Kwon et al., 2007). Two Arabidopsis chloroplastic MsrB proteins as well as rice (Oryza sativa) MsrB protein exhibit differential expression patterns in different tissues and in response to treatments with various stressors (Vieira Dos Santos et al., 2005). More recently, Laugier et al. (2010) reported that MsrB1 and MsrB2 mutants of Arabidopsis show reduced growth and development under environmental constraints such as high light levels or low temperature.

Evidence has accumulated that regulation of the oxidation/reduction states of Met in proteins is a novel molecular mechanism for the control of various cell functions (Levine et al., 2000; Hoshi and Heinemann, 2001; Moskovitz, 2005). Plants respond to pathogen attacks by oxidative “bursts,” which constitute the rapid production of ROS at the site of an attempted invasion (Apostol et al., 1989). In response to an incompatible pathogen attack, plants produce more ROS and coincidently decrease ROS-scavenging activities, which results in the accumulation of ROS and activation of the hypersensitive response (HR; Apel and Hirt, 2004). Without suppression of ROS-scavenging enzymes such as ascorbate peroxidase and catalase, ROS production at the apoplast alone does not induce HR in response to incompatible pathogen attacks (Mittler et al., 1999; Delledonne et al., 2001). These results indicate that the coordination between the suppression of ROS-scavenging enzymes and the accumulation of ROS is crucial for the onset of HR. Therefore, MsrB as an important ROS-modulating enzyme might be an important factor in plant defense by regulating the oxidation/reduction state of Met and the oxidative state of cells.

In this report, we describe the roles of pepper (Capsicum annuum) MsrB (CaMsrB2) in plant defense against pathogens using gain- and loss-of-function studies by overexpression of CaMsrB2 in tomato (Solanum lycopersicum) plants and suppression of CaMsrB2 by virus-induced gene silencing (VIGS) in pepper plants. To our knowledge, this work represents one of the most thorough studies demonstrating the roles of plant MsrB in defense against pathogens and oxidative stress.

RESULTS

Characterization and Localization of MsrB2 in Pepper

The isolated CaMsrB2 (National Center for Biotechnology Information [NCBI] accession no. EF144171) open reading frame encodes a full-length protein of 185 amino acid residues. We also isolated CaMsrB3 and CaMsrB1 (NCBI accession nos. EF144173 and EF144174, respectively) from the pepper EST sequence database (http://plant.pdrc.re.kr; Supplemental Fig. S1). A search of protein databases revealed that CaMsrB2 contains the typical putative four conserved motifs in a SelR domain, although plant MsrBs do not maintain selenium at the SelR domain (Fig. 1; Supplemental Fig. S2). The SelR domain, which is a conserved 121-amino acid region that is present in MsrBs of living organisms and is the catalytic region of MsrBs, exhibits a particularly high degree of similarity across different plant MsrBs. When comparing the SelR domain only, CaMsrB2 shares 76% amino acid identity with Arabidopsis MsrB (NCBI accession no. NP_567639). Multiple alignments of MsrB sequences reveal that CaMsrB2 has a conserved recycling (in the SGCGWPAF domain in box II) and a conserved catalytic (in the RxCxN motif in box IV) Cys residue that is involved in a redox process (Cys-119 and Cys-172, equivalent to Cys-63 and Cys-117 in Escherichia coli, respectively). Conserved Cys residues in positions 101, 104, 147, and 150 are also found organized into two CxxC motifs in MsrB boxes I and III (Fig. 1A) and would be involved in zinc fixation (Kumar et al., 2002; Rouhier et al., 2006). Interestingly, CaMsrB2 not only contains the putative N-terminal chloroplast transit peptide sequences (http://www.cbs.dtu.dk/services/TargetP/) but also has putative basic residues (42KRRFR46) as a nuclear localization signal (http://psort.nibb.ac.jp; Fig. 1A; Supplemental Fig. S1).

Figure 1.
Characterization of the CaMsrB2 gene. A, Alignment of MsrB amino acid sequences from pepper (CaMsrB2; GenBank accession no. EF144171), Arabidopsis (AtCaMsrB2; ...

The copy number of the CaMsrB2 gene in the chili pepper genome was estimated by DNA-blot analysis. When chili pepper cv Bukang genomic DNA was digested with three different restriction enzymes and hybridized with CaMsrB2 cDNA, the CaMsrB2 gene was estimated to be present as a single copy or at a low copy number (Fig. 1B). Although a restriction site for EcoRI within the CaMsrB2 cDNA was not detected (Supplemental Fig. S1), the double band pattern may result from endogenous restriction enzyme sites in the CaMsrB2 genomic DNA (Fig. 1B). The expression levels of the CaMsrB2 gene were investigated in different parts of the wild-type chili pepper plants. CaMsrB2 expression was highly detectable in the leaf, stem, and flower but detectable only at low levels in the root (Fig. 1C).

To investigate the cellular localization of CaMsrB2, we performed in vivo targeting experiments using constructs composed of CaMsrB2 fused to a soluble modified GFP (smGFP) as the fluorescent marker (David and Vierstra, 1996; Fig. 2A). Green fluorescence was detected in Nicotiana benthamiana protoplasts transfected with the CaMsrB2-smGFP construct or with 35S-smGFP. Protoplasts transfected with the CaMsrB2-smGFP construct emitted green fluorescence in the cytoplasm and nucleus (Fig. 2B), while protoplasts transfected with the 35S-smGFP construct showed GFP fluorescence only in the cytoplasm. To confirm the nuclear localization of CaMsrB2, we stained the nuclei with 4′,6-diamino-phenylindole (DAPI), and the CaMsrB2-SmGFP green fluorescence was found to overlap with DAPI fluorescence in the nuclei. These observations strongly indicate that CaMsrB2 is targeted to both the cytoplasm and nucleus (Supplemental Fig. S3).

Figure 2.
Subcellular localization of CaMsrB2. A, Construct map of the CaMsrB2-smGFP fusion protein. The CaMsrB2 coding region fused in-frame to the smGFP was inserted in pUC19 for in planta expression. ORF, Open reading frame. B, Location of the CaMsrB2-smGFP ...

Expression of CaMsrB2 in Response to Biotic Stress

To understand the function of CaMsrB2 in the defense response, the expression levels of CaMsrB2 were monitored in pepper plants treated with various pathogens and signal molecules. CaMsrB2 expression was detected in cv Early Calwonder-30R (ECW-30R; Bs3/Bs3) inoculated with Xanthomonas axonopodis pv vesicatoria (Xav) race 1 (avirulent Xav; AvrBs3/AvrBs3) and Xav race 3 (virulent Xav; avrbs3/avrbs3). ECW-30R pepper plants are susceptible to virulent Xav race 3 but resistant to avirulent Xav race 1; thus, either disease or HR is observed in response to infection by virulent Xav or avirulent Xav race 1, respectively (Kousik and Ritchie, 1996). Cultivar Bukang pepper plants, a nonhost for X. axonopodis pv Glys 8ra (Xag 8ra; Hwang et al., 1992), were also used for inducing HR following inoculation of Xag 8ra (Yi et al., 2004). In normal growth conditions, CaMsrB2 was constitutively expressed in ECW-30R. However, the expression gradually decreased upon infection with avirulent Xav race 1 and eventually was not detectable at 6 h after infection (Fig. 3A). However, ECW-30R showed a different CaMsrB2 expression profile in response to inoculation with Xav race 3. ECW-30R initially showed decreased expression of CaMsrB2 following infection by Xav race 3, as seen in the inoculation with avirulent Xav race 1. However, the expression level gradually recovered 18 h after Xav race 3 inoculation and attained its original level at 48 h post inoculation (Fig. 3A). In Bukang pepper plants, the expression of CaMsrB2 gradually decreased upon inoculation of Xag 8ra and remained at a low level (Fig. 3A). CaPR1, a positive marker for bacterial pathogen infection (Lee et al., 2002), was induced 6 h after the inoculation of Xag 8ra, and the expression gradually increased and reached a maximum at 24 h after inoculation (Fig. 3A). Mock inoculation of Bukang pepper plants with 10 mm MgCl2 moderately affected the expression of CaMsrB2 without inducing the expression of CaPR1 (Fig. 3A).

Figure 3.
Analysis of the expression patterns of CaMsrB2 and enzymatic activity of CaMsrB2. A, Expression of CaMsrB2 in ECW-30R pepper inoculated with Xav race 1 (incompatible) or race 3 (compatible) or with the nonhost pathogen Xag 8ra. Total RNA was extracted ...

Expression of CaMsrB2 in Response to Defense Signaling Molecules

To test the possible roles of defense-related signal molecules in the expression of CaMsrB2, chili pepper leaves were treated with defense signaling molecules such as methyl jasmonate (MJ), salicylic acid (SA), and ethephon. The expression of CaMsrB2 was suppressed by treatment with all these defense-related signaling molecules. MJ treatment significantly reduced the expression of CaMsrB2 at 1 h after treatment (Fig. 3B), while expression of CaMsrB2 gradually decreased with 5 mm SA treatment (Fig. 3B). Ethephon treatment also significantly lowered the expression of CaMsrB2 after only 0.5 h of treatment. The successful infiltration of MJ, SA, and ethephon was verified by recording the transcript levels of CaPinII, CaPR1, and CaACCoxidase as positive markers of MJ, SA, and ethephon treatment, respectively (Chung et al., 2007).

In addition, the enzymatic activity of the CaMsrB2 protein was determined using dabsyl-Met-sulfoxide (dabsyl-Met-SO) as a substrate. Dabsyl-Met-SO is a racemic mixture of dabsyl-Met-R-SO and dabsyl-Met-S-SO. CaMsrB2 and a CaMsrB2-mutant, in which the catalytic Cys-172 is changed to an Ala, were expressed with an N-terminal poly-His tag, and the protein was purified by affinity chromatography. The proteins were then assayed for enzymatic activity in converting dabsyl-Met-R-SO to dabsyl-Met. In the absence of proteins, dabsyl-Met-SO was separated into two green peaks, the S-form located at 27.7 min and the R-form located at 29.3 min. Addition of the purified CaMsrB2 mutant protein did not result in any difference to either the R- or the S-form peak compared with the peaks in the buffer control. In contrast, when the purified CaMsrB2 protein was added to dabsyl-Met-SO, the peak area for dabsyl-Met-R-SO was significantly decreased and a new peak for dabsyl-Met, the reduced form of dabsyl-Met-R-SO, appeared at 38.4 min. These results confirmed that the recombinant protein has MsrB activity (Supplemental Fig. S4).

Silencing CaMsrB2 in Pepper Plants

To understand the role of CaMsrB2 in pathogen defense response, the VIGS technique was applied to ECW-30R pepper plants in order to silence the CaMsrB2 gene. Two VIGS vectors, CaMsrB2-N containing the N-terminal region of CaMsrB2 and CaMsrB2-C containing the 3′-untranslated region (UTR) of CaMsrB2, were constructed (Fig. 4A, top). ECW-30R plants silenced with the CaMsrB2-N or -C construct showed retarded growth compared with control plants inoculated with a tobacco rattle virus (TRV) vector containing the GFP gene (Fig. 4A, bottom). CaMsrB2 expression was dramatically reduced in ECW-30R pepper plants silenced either with CaMsrB2-N or -C compared with the control plants (Fig. 4B). Semiquantitative reverse transcription (RT)-PCR was performed to determine the expression levels of CaMsrB1 and CaMsrB3 in CaMsrB2-N- or -C-silenced ECW-30R plants, as controls. The suppression of CaMsrB2 did not affect the expression level of CaMsrB1 or CaMsrB3 (Fig. 4C). To address the possible role of CaMsrB2 in incompatible and compatible pathogen attacks, we first detected the accumulation of H2O2 after infiltration (optical density at 600 nm [OD600] = 0.1–0.3) of incompatible pathogen Xav race 1 or compatible race 3 into the CaMsrB2-silenced ECW-30R using the 3,3′-diaminobenzidine (DAB) staining method (Minetti et al., 1994). The results revealed that production of H2O2 by Xav race 1 inoculation was stronger and more abundant than by Xav race 3 in the CaMsrB2-silenced plants (Supplemental Fig. S5).

Figure 4.
VIGS of the CaMsrB2 gene in ECW-30R pepper plant. A, Diagram of silencing constructs CaMsrB2-N (targeting the N terminus of CaMsrB2) and CaMsrB2-C (targeting the C terminus of CaMsrB2). The CaMsrB2-N and -C fragments were designed not to include the SelR-like ...

Accelerated Cell Death in CaMsrB2-Silenced Pepper Plants

Xav race 1 was infiltrated into the CaMsrB2-silenced ECW-30R plants to investigate the role of CaMsrB2 in HR. Yellow lesions, a typical symptom of HR, are induced by local cell death to limit the spread of pathogens (Lamb and Dixon, 1997). Visual inspection verified that this HR symptom was more severe in CaMsrB2-silenced plants than in the control plants 24 h after Xav race 1 inoculation (Fig. 5A). To determine the rate of cell death, trypan blue staining was performed on the leaves of ECW-30R plants inoculated with Xav race 1. The accumulation of blue color on leaf discs treated with a solution of trypan blue indicates dead cells. Blue spots were detected 24 h after Xav race 1 infiltration in the leaves of the CaMsrB2-silenced plants, and the most marked cell death was clearly detectable 48 h after Xav race 1 infiltration (Fig. 5B).

Figure 5.
Effects of silencing CaMsrB2 on HR. A, Cell death in the silenced plants. Leaves silenced with GFP (control), CaMsrB2-N, or CaMsrB2-C were infiltrated with Xav race 1. B, Cell death on the leaves was verified visually and by trypan blue staining. Dark ...

To understand the mechanism underlying the accelerated induction of HR in CaMsrB2-silenced plants, we detected O2· using nitroblue tetrazolium (NBT) staining. Production of insoluble blue-colored formazan complex (reduced NBT) indicates the existence of O2· (Doke, 1983). Between 3 and 12 h after Xav race 1 infiltration, accumulation of O2· was observed only in the CaMsrB2-N- and -C-silenced pepper leaves (Fig. 5C, left), suggesting that CaMsrB2-N- and -C-silenced plants respond to Xav race 1 by increasing the amount of O2·. In CaMsrB2-N-silenced plants, the accumulation of O2· reached the highest amount (2.2 μm g−1 fresh weight h−1) at 6 h after Xav race 1 infection and gradually decreased and stayed almost at the same concentrations as those in the control plants 24 h post infection. CaMsrB2-C-silenced plants showed a similar pattern of O2· accumulation as the CaMsrB2-N-silenced plants, except that the highest accumulation of O2· was at 12 h after infiltration of Xav race 1 (Fig. 5C, right).

The levels of H2O2 in ECW-30R plants inoculated with Xav race 1 were analyzed using the DAB staining method, which specifically detects only H2O2 among ROS (Minetti et al., 1994). When Xav race 1 was inoculated into the controls and the CaMsrB2-silenced plants, both plant types responded to the infection by increasing the amounts of H2O2 (Fig. 5D, left). However, the accumulation of H2O2 was higher in the CaMsrB2-silenced plants than in the control plants. Specifically, the CaMsrB2-N-silenced plants contained 16 μm g−1 fresh weight H2O2 compared with 10 μm g−1 fresh weight H2O2 in the control plants at 12 h after infection, and the CaMsrB2-C-silenced plants contained 14 μm g−1 fresh weight H2O2 compared with 10 μm g−1 fresh weight H2O2 in the control plants at 24 h after infection (Fig. 5D, right).

Increased Susceptibility to Virulent Xav in CaMsrB2-Silenced Plants

To analyze the effect of CaMsrB2 silencing on the basal defense response, leaves of the CaMsrB2-silenced ECW-30R plants were inoculated with virulent Xav, which causes black spot wilt disease (Kousik and Ritchie, 1996). At 4 d after inoculation (DAI), the numbers of virulent Xav were significantly higher in CaMsrB2-C-silenced plants than in the control plants, and at 6 DAI, the numbers of virulent Xav were significantly higher in CaMsrB2-N- and -C-silenced plants than in the control plants (Fig. 6A). Semiquantitative RT-PCR data clearly showed that the CaMsrB2-silenced plants exhibited reduced expression of CaMsrB2 compared with the control plants (Fig. 6B). All these data suggested that silencing of CaMsrB2 in pepper plants resulted in increased susceptibility to virulent Xav. The underlying mechanisms for the reduced resistance in the CaMrsB2-N- and -C-silenced pepper plants were further investigated by measuring the expression levels of pathogenesis-related proteins 4 and 10 (CaPR4 and CaPR10, respectively). The expression level of CaPR10 in the control was significantly higher than that in the CaMsrB2-N-silenced plants at 6 DAI and significantly higher than that in the CaMsrB2-C-silenced plants at 4 DAI. The transcript accumulation of CaPR4 in the control plants was more rapid than that in the CaMsrB2-N- and -C-silenced plants. The CaMsrB2-N-silenced plants accumulated less CaPR4 at 6 DAI than the control plants, while CaMsrB2-C-silenced plants accumulated CaPR4 at 4 DAI, 2 d slower when compared with the control plants. The expression levels of several antioxidant enzymes were also monitored, and as a result, the expression of a cytosolic copper/zinc (Cu,Zn) superoxide dismutase (CaSOD) was found to be higher in the control plants than in the CaMsrB2-silenced plants at 4 and 6 DAI. These results indicate that silencing of CaMsrB2 in pepper plants affects the levels of defense-related gene transcripts and ROS-scavenging enzymes.

Figure 6.
Enhanced susceptibility to Xav race 3 in CaMsrB2-silenced pepper plant. A, Bacterial growth in the silenced plants. Leaves of GFP (control), CaMsrB2-N-, or CaMsrB2-C-silenced plants were infiltrated with Xav race 3 (approximately 6 × 104 cfu mL ...

Overexpression of CaMsrB2 in Tomato Confers Resistance to Phytophthora Pathogens

Because the knockdown of CaMsrB2 reduced the defense response, we reasoned that its overexpression might enhance disease resistance. In order to quantify the effect of CaMsrB2 on disease resistance responses in planta, we generated transgenic tomato plants that constitutively express CaMsrB2 under the control of the cauliflower mosaic virus 35S promoter using Agrobacterium tumefaciens-mediated transformation. Twelve CaMsrB2-transgenic lines showed strong expression and were selected. The CaMsrB2-expressing transgenic tomato plants accumulated significantly more fresh weight than the vector-only-transformed control plants (data not shown). This finding supposed that the overexpression of CaMsrB2 affected the growth enhancement in tomato transgenic plants. In the T3 generation, three transgenic lines that stably expressed CaMsrB2 were selected for further analysis.

We studied the role of the CaMsrB2 gene in defense using the CaMsrB2-transgenic tomato plants infected with Phytophthora infestans and Phytophthora capsici, which induce tomato or pepper late and foliar blight disease, respectively, and which are the most devastating members of the oomycete genus Phytophthora, respectively (Fry and Goodwin, 1997; Housbeck and Lamour, 2004). Disease symptoms were visible in both the control and CaMsrB2-transgenic tomato plants within 3 d of P. capsici (Fig. 7) and P. infestans (Supplemental Fig. S6) infection. However, the area of infection was significantly smaller in all T3 CaMsrB2-transgenic lines (lines 16, 17, and 18) compared with the control plants (Fig. 7A; Supplemental Fig. S6), and this could be correlated with the reduced fungal colonization as observed by trypan blue staining analysis (Fig. 7A). At 4 DAI, in the transgenic tomato lines, visible disease symptoms were observed only in 26.7% to 53.3% of the total leaf area, a significantly smaller area compared with the 85.0% of the total leaf area observed in the control plants (Fig. 7B).

Figure 7.
CaMsrB2 expression in tomato is more resistant to Phytophthora. A, Typical disease symptoms after inoculation of tomato leaves with P. capsici. Four days after inoculation, leaves were trypan blue stained to visualize disease development. B, Leaf area ...

In addition, the in planta colonization of Phytophthora was determined by measuring the expression of the P. capsici EF1α gene (for elongation factor 1α), a constitutively expressed gene, in order to calculate the amount of Phytophthora hyphae biomass in the infected tomato leaves. At 0, 1, 3, and 4 DAI, semiquantitative RT-PCR analysis revealed that levels of PcEF1α transcripts were significantly lower in the transgenic tomato lines than in the control plants. The tomato EF1α gene (LeEF1α) was used to confirm equal amounts of RNA loading among samples and for internal control of RNA quality (Fig. 7C). When semiquantitative RT-PCR analysis were performed to determine the amounts of LeEF1α transcript in CaMsrB2-transgenic plants, PcEF1α transcript levels were reduced by 25% to 58.5% when compared with the control plants (Fig. 7E). This may result from a loss of viable tissue during the progress of the disease.

The total peptide Msr activity in tomato transgenic leaf extracts was investigated by using dabsyl-Met-SO as a synthetic substrate (see “Materials and Methods”). Under normal conditions, all of the three selected CaMsrB2-transgenic tomato lines exhibited significantly enhanced peptide MsrB activities in their leaf extracts (Fig. 7D, left part). Under pathogen inoculation, the pMBP1-transgenic plants display a total peptide Msr activity of 30.96 pmol Met min−1 mg−1 protein, slightly higher than that measured under normal conditions (pMBP1). The activities in CaMsrB2-transgenic tomato plants (lines 16, 17, and 18) are also significantly increased by 33.8%, 29.2%, and 23.7%, respectively (Fig. 7D, right part). Therefore, these results indicate that the CaMsrB2 enzymes are an important part of the leaf peptide Msr capacity for defense responses in plants.

Effects of Overexpressing CaMsrB2 on H2O2 Production and Defense Gene Expression

In order to quantify the effects of CaMsrB2 overexpression in transgenic tomato on disease-associated cell death (DCD) and the level of H2O2 production, three different T3 CaMsrB2-transgenic plants were inoculated with P. capsici and the DAB staining method was employed to measure the amounts of H2O2. Disease symptoms were observed in both the control and CaMsrB2-transgenic tomato plants by 2 DAI of P. capsici and were significantly less marked in all the CaMsrB2-transgenic lines compared with the control plants (Fig. 8). The histological analyses revealed that P. capsici inoculation induced H2O2 production in leaves of both control and CaMsrB2-transgenic plants. However, a much reduced response was observed in CaMsrB2-transgenic leaves (Fig. 8, A and C). Interestingly, H2O2 production was restricted in the inoculated CaMsrB2-transgenic leaves but not in control leaves (Fig. 8A, right). The appearance of cell death in P. capsici-infected tomato leaves showed a similar pattern as the distribution of H2O2 (Figs. 7 and and8).8). These results demonstrate that suppression of cell death in CaMsrB2-transgenic plants is closely associated with the generation of H2O2.

Figure 8.
Effects of overexpressing CaMsrB2 on the Phytophthora blight disease. Leaves of CaMsrB2-transgenic tomato were inoculated with P. capsici zoospores (5 × 105 zoospores mL−1). A, Cell death in CaMsrB2-silenced plants. Cell death on the leaves ...

The effects of CaMsrB2-transgenic tomato plants on defense-related gene expression were also determined by quantitative RT-PCR analysis using specific primer sets (Supplemental Table S1) for several tomato defense-related genes and antioxidant genes that are known to be expressed during plant response to pathogens or oxidative stress (Lamb and Dixon, 1997). The overexpression of CaMsrB2 in tomato increased the transcript levels of LeP1 (for pathogenesis-related P14), LePR-1b, and LeP23 (osmotin-like protein; Fig. 8B). However, using RT-PCR, we did not detect a difference in the expression levels of several antioxidant enzymes in CaMsrB2-overexpressing tomato leaves (data not shown). For further examination of the roles of CaMsrB2 in transgenic tomato plants, we analyzed global gene expression using a tomato cDNA microarray. The up-regulation of several antioxidant genes in overexpressing CaMsrB2 tomato plants was detected (Supplemental Table S2). These results demonstrate that CaMsrB2 is associated with the coordination between the accumulation of H2O2 and cell death from pathogen attacks. Taken together, these results suggest that a ROS-scavenging enzyme, CaMsrB2, might be an important factor in plant defense by regulating the oxidative state of plant cells.

Overexpression of CaMsrB2 in Tomato Increased the Tolerance to Oxidative Stress

The CaMsrB2-transgenic lines were tested for tolerance to oxidative stress. When the tomato leaf discs of transgenic lines (lines 16, 17, and 18) or of the control were submerged in MV and exposed to light for 2 d, the chlorinated regions around the leaf discs were significantly more distinct in the control leaf discs (Fig. 9A, top). In addition to visual detection, significantly higher levels of electrolyte leakage were recorded in the control than in the transgenic lines (Fig. 9A, bottom). CaMsrB2-transgenic and control tomato leaf discs were also submerged in 0, 0.4, or 0.8 m H2O2, and chlorophyll levels were recorded. Although there were differences in the amounts of chlorophyll among the transgenic lines (Fig. 9B, top), significantly higher levels of chlorophyll remained in the CaMsrB2-transgenic leaf discs even after treatment with 0.4 or 0.8 m H2O2. These results indicated that overexpression of CaMsrB2 in tomato plants enhances the tolerance to oxidative stress.

Figure 9.
Ectopic expression of CaMsrB2 and tolerance levels to oxidative stresses. A, Electrolyte leakage of CaMsrB2 transgenic tomato leaves following treatment with MV. Electrolyte leakage was measured at 2 d after treatment of 0.005 mm MV. Photographs were ...

DISCUSSION

ROS can act as signaling molecules that regulate plant development, stress adaptation, and HR to pathogen (Apel and Hirt, 2004). Excessively produced ROS can cause damage to DNA, lipids, and proteins, especially those with the sulfur-containing amino acid Met (Gao et al., 1998). To repair Met-oxidized proteins, Msr enzymes catalyze the reduction of MetSO to Met (Stadtman et al., 2005). Recent studies using mutated Msr genes in Arabidopsis and rice plants have suggested significant roles of Msr proteins in oxidative stress or plant defense responses (Vieira Dos Santos et al., 2005; Kwon et al., 2007; Laugier et al., 2010); however, the function of plant Msr in defense response to pathogens and oxidative stress is not fully understood. Here, we identified a new chili pepper CaMsrB2, which has a reduction capacity that is specific for the oxidized Met-R-SO form of Met. CaMsrB2 was down-regulated by pathogen infection and by classical defense-related signal molecules (Fig. 3). Furthermore, the results from silencing or overexpression of CaMsrB2 indicate that CaMsrB2 regulates pathogen defense responses and oxidative stress by controlling ROS accumulation and reduction, respectively.

All eukaryotic and most prokaryotic organisms have MsrA and MsrB, which reduce Met-S-SO and Met-R-SO, respectively, in proteins (for review, see Zhang and Weissbach, 2008). MsrA and MsrB are not structurally related but catalyze a similar reaction using as substrate the diastereoisomers of Met-O. It is possible that the Cys-containing motif RxCxN at the C terminus of MsrB, including CaMsrB2, is essential for the catalytic activity (Lowther et al., 2002; Rouhier et al., 2006). The MsrB genes constitute a multigene family with nine different members in Arabidopsis (Vieira Dos Santos et al., 2005; Tarrago et al., 2009), but only three MsrB genes have been identified in chili pepper (Supplemental Figs. S1 and S2). As observed in Arabidopsis and pepper, most plants contain more than one copy of the MsrB gene, whereas several eukaryotes, such as Drosophila melanogaster, Caenorhabditis elegans, and Saccharomyces cerevisiae, contain only one copy of MsrB (Kryukov et al., 2002). The multiple copies of MsrB in plants suggest that plants require various types of MsrB for diverse physiological functions. In mammalian systems, overexpression of MsrA in human T-lymphocyte cells protects the cells against oxidative stress (Moskovitz et al., 1998), whereas MsrA knockout mice are more sensitive to hyperbaric oxygen (Moskovitz et al., 2001). MsrA-overexpressing flies are more resistant to paraquat (MV; Ruan et al., 2002). In plants, Arabidopsis endoplasmic reticulum-localized MsrB3 or plastidic MsrA in chloroplasts was shown to play protective roles against oxidative damage such as MV treatment, which produces O2· by catalyzing the transfer of electrons from PSI to molecular oxygen (Romero et al., 2004; Kwon et al., 2007). When leaf discs of CaMsrB2-overexpressing tomato plants were immersed in a solution of MV or H2O2, the tomato leaves showed increased resistance to the oxidative stress. These data emphasize again that CaMsrB2 is an important antioxidant enzyme protecting cells from oxidative stress. Furthermore, in this study, the overexpression of CaMsrB2 in tomato plants was found to result in increased growth, while the silencing of CaMsrB2 in pepper plants resulted in retarded growth. The overexpression of CaMsrB2 enhanced tolerance to oxidative stress, which in turn may contribute to growth enhancement in the transgenic plants.

We also found that CaMsrB2 is an essential component regulating pathogen defense in plants. A previous report showed that MsrA and MsrB genes are expressed differentially during compatible or incompatible interactions resulting from infection by different races of the rust fungus Melampsora laricipopulina in poplar (Populus species) trees (Vieira Dos Santos et al., 2005). Pepper plants showed down-regulated CaMsrB2 expression in response to compatible and incompatible pathogen attack. In particular, pepper plants undergoing incompatible interactions displayed significantly decreased CaMsrB2 expression, and this reached an undetectable level at 6 h after the infection. CaMsrB2 expression was also down-regulated by major defense-inducing chemicals such as SA, MJ, and ethylene, suggesting that CaMsrB2 actively responds to the defense signals.

The generation of ROS is closely related to plant defense responses against pathogen attack, especially HR, and is essential for the establishment of plant immunity (Torres et al., 2002; van Loon et al., 2006). One of the most active defense responses to pathogen attacks is the oxidative burst, which leads to the transient production and accumulation of large amounts of ROS such as H2O2 and O2· (Lamb and Dixon, 1997). We found that CaMsrB2 regulates the production of ROS during defense responses as well as the induction of HR from pathogen attack. Accumulation of ROS such as O2· and H2O2 plays a central role in HR induced by an attack of incompatible pathogens (Levine et al., 1994). In the infection stage, first, ROS are produced via the enhanced enzymatic activity of plasma membrane-bound NADPH oxidase, cell wall-bound peroxidase, and apoplastic amine oxidases (Torres et al., 2005; Choi et al., 2007). Second, the HR is activated by induction of SOD, which converts O2· to H2O2, and antioxidant enzymes are induced by overproduction of O2· but not H2O2. These results indicate that the HR is triggered by H2O2 rather than O2· (Delledonne et al., 2001; Yi et al., 2010). Third, Beers and McDowell (2001) demonstrated that O2· can either activate the HR, providing H2O2, or suppress the HR by inducing antioxidant enzymes and by removing nitric oxide as molecules triggering cell death.

We showed that higher amounts of ROS in CaMsrB2-silenced pepper plants, especially O2· between 3 and 12 h after infection with avirulent Xav, were accumulated in a shorter time. Furthermore, we observed that CaMsrB2-silenced plants show rapid and high-level DAB staining, as a marker of H2O2 accumulation, 12 to 24 h after challenge with the avirulent Xav (Figs. 4 and and5).5). Therefore, the greater accumulation of ROS can accelerate HR cell death in the infection by avirulent Xav, which is an incompatible interaction in pepper. Likewise, in response to an incompatible pathogen attack, plants produce more ROS and coincidently decrease the ROS-scavenging activities, which results in the accumulation of ROS and activation of the HR (Mittler et al., 1999). In leukemia cells, overexpression of mitochondrial CaMsrB2 delayed apoptosis by preservation of mitochondrial integrity, resulting from a decrease in the levels of ROS (Cabreiro et al., 2008). As shown in Figure 3, the overall down-regulation of CaMsrB2 in ECW-30R during an attack of the incompatible pathogen avirulent Xav could be interpreted as a mechanism in ECW-30R pepper plants to intentionally increase the amounts of ROS to induce HR. There may not be any need to repair the oxidized Met-R-SO in HR-induced pepper plants, because they are in the process of actively dying; therefore, there is no reason for CaMsrB2 expression.

The expression pattern of CaMsrB2 in pepper plants differs in compatible interactions when compared with incompatible interactions. The expression levels of CaMsrB2 were decreased in pepper plants at the beginning of infection with virulent Xav; however, CaMsrB2 expression had recovered to its original level by 48 h after the infection. We also found that CaMsrB2-silenced plants showed a low intensity of DAB staining after challenge with the virulent Xav. Suppression of CaMsrB2 resulted in increased susceptibility to the virulent Xav. The enhanced pathogen growth in the CaMsrB2-silenced pepper plants may result from easier cellular disruption caused by partial damage from the ROS-scavenging system. It was also found that the expression levels of several antioxidant enzymes, including CaSOD, were lower in CaMsrB2-silenced plants than in the control plants. Furthermore, the expression of CaMsrB2 protein might have a role in the regulation of the defense-related genes CaPR4 and CaPR10. PR4 and PR10, a hevein-like protein and a ribonuclease, respectively, play important roles in pathogen defense (van Loon et al., 2006). CaMsrB2-silenced pepper plants showed reduced expression of CaPR4 and CaPR10, and down-regulation of these genes may partially explain why virulent Xav grew faster in CaMsrB2-N- and -C-silenced ECW-30R pepper plants. We also showed that CaMsrB2 overexpression in tomato plants induced defense-related tomato genes such as LeP1, LePR1b, and LeP23. In addition, up- or down-regulation of plastidic and several cytosolic antioxidant genes, or defense-related genes, in CaMrsB1-overexpressing tomato plants (Supplemental Table S2) implies that there is cross talk among different organelles, including the cytosol, nucleus, chloroplast, and endoplasmic reticulum.

It has been reported that accumulation of ROS is usually associated with successful disease resistance and HR (Torres et al., 2002; Choi et al., 2007). Furthermore, H2O2 may contribute to cell wall reinforcement, thus limiting the pathogen spread, and can also directly participate in pathogen destruction (Lamb and Dixon, 1997). CaMsrB2-overexpressing tomato plants show enhanced growth, which may trigger increased resistance to the oomycete pathogen Phytophthora through thicker cell walls. Overexpression of the CaMsrB2 gene also conferred enhanced disease resistance accompanied by cell death, H2O2 accumulation, and PR gene induction following P. capsici infection (Figs. 7 and and8).8). In CaMsrB2-transgenic tomato plants, the amount of H2O2 was reduced during Phytophthora infection and disease resistance was enhanced (Fig. 8A). Interestingly, H2O2 accumulation and cell death were highly condensed and restricted to the region of pathogen invasion in CaMsrB2-transgenic leaves. In contrast, accumulation of H2O2 was widely distributed in the entire leaf area of control plants, which were more susceptible and showed increased cell death from P. capsici infection. Likewise, this accumulation of ROS seemed to be induced by plant cell death caused by DCD or HR. These results may indicate that CaMsrB2 expression and H2O2 accumulation are involved in defense against P. capsici, but the defense and the concentration of H2O2 in entire leaves seem not to be correlated with DCD.

Our data strongly suggest that the newly characterized CaMsrB2 gene has novel defensive roles in pathogen attack via regulation of the cellular levels of ROS and defense-related genes. The oxidation/reduction state of Met in cellular proteins can regulate cellular functions in most living systems (Hoshi and Heinemann, 2001). The target protein of CaMsrB2 function during pathogen challenge is not yet identified; however, understanding the oxidation/reduction state of Met in the target protein will provide greater insights into the role of MsrB protein in pathogen and oxidative stress defense in plants.

MATERIALS AND METHODS

Bacterial Pathogen Inoculation

Chili pepper (Capsicum annuum) cv Bukang and cv ECW-30R seeds were grown on Murashige and Skoog (MS) medium for 2 weeks, transferred to pots, and then grown in a growth chamber at 24°C for 4 weeks. The inoculated bacterial pathogens included Xanthomonas axonopodis pv Glys 8ra, a soybean pustule pathogen (Hwang et al., 1992), and Xanthomonas axonopodis pv vesicatoria race 1 and race 3 (avirulent Xav race 1 and virulent race 3), pepper bacterial spot pathogens (Kousik and Ritchie, 1996). Bacteria grown in yeast extract-dextrose-CaCO3 medium at 30°C were harvested and resuspended in 10 mm MgCl2 to a concentration of approximately 4 × 108 colony-forming units (cfu) mL−1 or to OD600 = 0.05. Bacterial suspensions were infiltrated into leaf mesophyll tissues of 4-week-old pepper plants using a 1-mL needleless syringe.

DNA and RNA Gel Analysis

Genomic DNA was isolated from chili pepper leaves as described by Lee et al. (2002). Genomic DNA (20 μg) was digested with EcoRI, HindIII, or XbaI. The digested DNA was separated by electrophoresis on a 0.7% agarose gel, denatured, and blotted onto a Nytran membrane (Amersham Pharmacia). DNA-blot hybridization was performed by using the 3′-UTR of CaMsrB2 cDNA as the probe labeled with [32P]dCTP. For RNA gel-blot analysis, total RNA (20 μg) was separated on a formaldehyde-containing agarose gel and transferred onto a Nytran membrane (Amersham Pharmacia). Each cDNA probe was labeled with [32P]dCTP using the Prime-a-Gene System (Promega).

Cellular Localization of CaMsrB2

To determine the subcellular localization of CaMsrB2 protein, the CaMsrB2-smGFP fusion protein was used. The full-length open reading frame of CaMsrB2 (residues 1–185 amino acids) without the termination codon was prepared by PCR using CaMsrB2 cDNA as a template and a primer set (forward primer 5′-GGATCCATGGTTCTCAGATTCTC-3′ and reverse primer 5′-GGATCCCGGTTTGCCGGTGTAAACTTGAGGGAAATAC-3′). The underlined nucleotides contain the BamHI restriction site. The C-terminal region of the PCR-amplified CaMsrB2 fragment was fused to the N-terminal region of the smGFP expression vector (David and Vierstra, 1996). The 35S-smGFP vector without CaMsrB2 was used as a control. For transient expression analysis, 4 μg of 35S-CaMsrB2-smGFP plasmid or 35S-smGFP plasmid was introduced into the protoplasts of Nicotiana benthamiana using polyethylene glycol-mediated transformation (Cho et al., 2004). Expression of the fusion constructs was monitored at 24 h after transformation by a confocal laser scanning microscope (Carl Zeiss LSM 510). The filter sets used were BP 505 to 530 (excitation, 488 nm; emission, 505–530 nm), BP 385 to 470 (excitation, 314 nm; emission, 385–470 nm), and LP 650 (excitation, 488 nm; emission, 650 nm; Carl Zeiss) for GFP, DAPI, and autofluorescence of chlorophyll, respectively.

VIGS of CaMsrB2

A stock solution of Agrobacterium tumefaciens strain GV2260 for VIGS was prepared as described by Liu et al. (2002). CaMsrB2-N (targeting the N terminus of CaMsrB2) and CaMsrB2-C (targeting the C-terminal UTR of CaMsrB2) were prepared by PCR with specific primers (Supplemental Table S1). Agrobacterium was transformed with TRV2 plasmids containing CaPDS (Chung et al., 2004), GFP, CaMsrB2-N, or CaMsrB2-C. Single colonies of Agrobacterium transformed with these genes were grown in 5 mL of YEP (10 g yeast extract, 10 g peptone, 5 g NaCl) medium containing rifampicin (50 μg mL−1) and kanamycin (50 μg mL−1) overnight. The transformed Agrobacterium was harvested by centrifugation at 3 × 103 rpm for 15 min at 20°C, resuspended to 0.5 A600 in 10 mm MgCl2 with 100 μm acetosyringone, and exposed at 22°C with shaking for 4 h. Agrobacterium with TRV1 and Agrobacterium with TRV2:GFP, TRV2:CaPDS, TRV2:CaMsrB2-N, or TRV2:CaMsrB2-C were mixed at a 1:1 ratio and then infiltrated into the cotyledons of 7-d-old ECW-30R seedlings using a 1-mL needleless syringe. Each construct was used for silencing CaMsrB2 in 20 seedlings, and after 20 d of the VIGS treatment, ECW-30R pepper plants were inoculated with avirulent Xav (at OD600 = 0.05 or 0.1) or virulent Xav race 3 (approximately 6 × 104 to approximately 6 × 108 cfu mL−1). All experiments were repeated at least twice.

Agrobacterium-Mediated Transformation of Tomato

Seeds of tomato (Solanum lycopersicum ‘Micro Tom’) were obtained from the National Horticultural Research Institute of Korea. Surface sterilization was done by immersing the seeds in 1% (v/v) NaOCl at room temperature for 10 min, followed by washing the seeds with sterilized distilled water for 5 min. Seeds were then germinated on MS agar medium and kept in a plant growth chamber under a 16-h photoperiod at 25°C for 2 weeks before being used for transformation.

CaMsrB2 full-length cDNA was cloned into the pMBP1 plant gene expression vector. The recombinant vector was transferred to the Agrobacterium strain LBA 4404 by the freeze-thaw method described by An et al. (1987). Cotyledons from 2-week-old tomato seedlings germinated on MS medium were used for cocultivation with Agrobacterium to generate transgenic plants, according to the method of van Roekel et al. (1993). Kanamycin-selected transgenic plants were grown in a greenhouse, and then transgenic plants were further selected by measuring the expression of the nopaline synthase terminator by PCR with a primer set (Supplemental Table S1).

Evaluation of CaMsrB2-Transgenic Tomato Resistance to Phytophthora

Four-week-old T3 tomato (cv Micro Tom) plants were inoculated with the zoospores of Phytophthora capsici isolate KACC40470 (Korean Agriculture Culture Collection) and Phytophthora infestans (provided by Korea Research Institute of Chemical Technology). For inoculum, P. capsici was grown at 28°C (for P. infestans, at 20 °C) under a 16-h photoperiod for 3 d, and the sporangia were harvested in sterile water and stimulated to release zoospores by incubation at 4°C for 2 to 3 h. After filtration through muslin, the numbers of sporangia were counted with a light microscope, adjusted to 3 × 104 sporangia mL–1, and sprayed onto fully expanded tomato leaves. The inoculated plants were kept in a humidity chamber for the first 48 h at 28°C (for P. infestans, at 20°C) and then incubated at 28°C (for P. infestans, at 20°C) with 70% humidity for up to 4 d. Six independent plants from each transgenic line were infected, and disease symptoms appeared within 2 to 3 DAI. The index of Phytophthora infection was determined visually based on the necrotic and curled leaf area. The experiments were repeated at least twice.

CaMsrB2 Enzyme Assay

The enzymatic activity of CaMsrB2 recombinant protein was monitored by measuring the reduction of the synthetic substrate, dabsyl-Met-SO. The reduction of Met-SO to Met was assayed using dabsyl-Met-SO, the substrate described by Moskovitz et al. (2002). Dabsyl-Met and dabsyl-Met-R,S-SO were synthesized according to the description of Minetti et al. (1994). The reaction mixture, containing purified CaMsrB2 in 15 mm HEPES, pH 8.0, 10 mm MgCl2, 30 mm KCl, 20 mm 1,4-ditholerythritol (DTE), and 0.5 mm dabsyl-Met-SO in a final volume of 100 μL, was incubated for 1 h at 37°C. The reaction was stopped by adding 450 μL of 29 mm ethanol:acetate (50:50, v/v) buffer, pH 4.16, to the 50-μL reaction mixture. After centrifugation at 12,000g for 30 min at 4°C, supernatants were loaded into a C18 reverse-phase column (Symmetry C18 5 μm, 3 × 250 mm; Waters). Solvent A (29 mm acetate buffer, pH 4.16) and solvent B (acetonitrile) were used for the HPLC. The detector wavelength was set at 436 nm for absorbance reading. The program used for the HPLC assays was carried out according to the method described by Vieira Dos Santos et al. (2005).

Total Peptide Msr Activity Assay and Reverse-Phase HPLC of Dabsyl-Met

Total peptide Met-SO reductase activity in CaMsrB2-transgenic tomato plant leaf extracts was assayed by monitoring the reduction of the synthetic substrate, dabsyl-Met-SO, in the presence of DTE as described in previous papers (Vieira Dos Santos et al., 2005; Laugier et al., 2010). Tomato leaf samples were blended in liquid nitrogen, and the powder was resuspended in 15 mm HEPES, pH 8.0, 10 mm MgCl2, 30 mm KCl, and 1 mm phenylmethylsulfonyl fluoride and centrifuged at 15,000g for 30 min at 4°C. The supernatant was collected and the total protein content was determined using the Bradford protein assay (Bradford, 1976). The reaction mixture, containing 500 or 1,000 μg of total proteins in 15 mm HEPES, pH 8.0, 10 mm MgCl2, 30 mm KCl, 20 mm DTE, and 0.5 mm dabsyl-Met-SO in a final volume of 100 μL, was incubated for 1 h at 37°C. The reaction was stopped by adding 900 μL of 29 mm ethanol:acetate (50:50, v/v) buffer, pH 4.16, to the 100-μL reaction mixture. After centrifugation at 12,000g for 30 min at 4°C, 20 μL of supernatants was loaded on a C18 reverse-phase column.

For reverse-phase HPLC analysis, the method was modified according to the description of Minetti et al. (1994). Solvent A (29 mm acetate buffer, pH 4.16) and solvent B (acetonitrile) were used for the HPLC analysis. The flow rate was 1 mL min−1 at 25°C. The detector wavelength was set at 436 nm for absorbance reading (Thermo Electron Surveyor PDA Plus Detector). The column utilized was a J’sphere ODS-H80 4 μm, 250 × 4.6 mm (YMC Co.) The program used for the HPLC assays was carried out according to the modified method described by Vieira Dos Santos et al. (2005). Briefly, it starts with 28% solvent B for 30 min, up to 100% B in 0.1 min, at 100% B for 9.9 min, down to 28% B in 0.1 min, and equilibration at 28% B for 9.9 min. The whole program lasts 50 min. In these conditions, dabsyl-Met-S-SO, dabsyl-Met-R-SO, and dabsyl-Met elute at 21.8, 22.9, and 33.5 min, respectively.

Electrolyte Leakage and Chlorophyll Content Analysis

For the electrolyte leakage test, 4-week-old tomato leaf discs of transgenic lines (lines 16, 17, and 18) and the control were submerged in 0.005 mm MV (Sigma) and exposed to light for 2 d. Electrolyte leakage measurements were performed as described by Warren et al. (1996). After treatment with H2O2 (0, 0.4, and 0.8 m), chlorophyll was extracted from transgenic or nontransgenic tomato leaves according to the method of Hu et al. (2005). The absorbance of chlorophyll extracts was measured at 663 and 645 nm, and then the chlorophyll content was calculated by the method of Lichtenthaler (1987).

Trypan Blue Staining

We used the trypan blue staining method to detect dead tomato cells or Phytophthora mycelia as described by Roetschi et al. (2001). First, we prepared the trypan blue stock solution (10 g of phenol, 10 mL of glycerol, 10 mL of lactic acid, 10 mL of water, and 0.02 g of trypan blue). The working solution was then prepared by diluting the stock solution with ethanol (96%, 1:2 [v/v]). Infected tomato leaf tissues were transferred into a 50-mL Falcon tube with a lid and covered with diluted trypan blue solution. The Falcon tubes were placed in a heated water bath, and the staining solution was boiled for 2 min. The tomato leaves were left overnight in the trypan blue staining solution and destained overnight in chloral hydrate solution (2.5 g mL−1).

RT-PCR Analysis

Total RNA samples were extracted from pepper using TRI reagent according to the manufacturer’s instructions (Invitrogen). Northern-blot probes such as CaPR1, CaPinII, and CaACC oxidase were selected from cDNA probes previously prepared in our laboratory (Lee et al., 2001; Yi et al., 2004; Chung et al., 2007). RT-PCR was performed to detect the endogenous levels of several genes using the primer sets listed in Supplemental Table S1. Time courses of P. capsici infection of CaMsrB2-transgenic tomato leaves were performed using zoospore spray inoculations (2 × 104 sporangia mL–1). Total RNA isolated from infected leaves of tomato, 1, 3, or 4 DAI, from noninfected leaves (0 DAI), and genomic DNA isolated from P. capsici mycelium grown in synthetic medium was amplified with primer sets from the two genes. Infected leaves were harvested and frozen in liquid nitrogen for immediate use or stored at −80°C for RNA extraction. First-strand cDNA was synthesized using 2 μg of total RNA, oligo(dT) primer, and M-MLV RT according to the manufacturer’s instructions (Invitrogen). The oligonucleotides used to amplify P. capsici PcEF1α (NCBI GenBank accession no. EU080853) are listed in Supplemental Table S1. The expression of the PcEF1α gene was controlled with a primer pair specific for the constitutively expressed tomato EF1α gene (Shewmaker et al., 1990; Supplemental Table S1). RT-PCR products were separated on 1.2% agarose gels and stained with ethidium bromide. Tomato LeEF1α expression was used to normalize the expression value in each sample, and relative mRNA expression values were determined by using the ImageJ program (http://rsb.info.nih.gov/ij/).

NBT Staining for Detection of O2·

Staining of NBT (Sigma-Aldrich) was used to detect the amounts of O2· (Doke, 1983). ECW-30R pepper plants were inoculated with avirulent Xav, and 0.5-cm-diameter leaf discs were acquired. Three leaf discs were immersed in 3 mL of 0.01 m potassium phosphate buffer (pH 7.8) containing 0.05% (w/v) NBT and 10 mm NaN3 for 1 h. After removing the leaf discs, the reaction solution was boiled at 85 °C for 15 min and cooled on ice. The oxidized NBT was measured at A580.

DAB Staining for H2O2 Detection

The levels of H2O2 were measured by a modified method of Capaldi and Taylor (1983). Leaf discs infiltrated with avirulent Xav were ground in 5% (w/v) TCA on ice and centrifuged at 4°C for 10 min at 15,000g. The supernatant was collected, adjusted to pH 3.6 with 4 n KOH, and used for the H2O2 assay. The supernatant (100 μL) was added to 50 μL of 3.4 mm 3-methylbenzothiazoline hydrazone (Sigma-Aldrich), and 250 μL of dissolved horseradish peroxidase (0.9 units mL−1) in 0.2 m sodium acetate (pH 3.6) was added to the mixture. After 2 min, 700 μL of 1 n HCl was added, and after 15 min, the A630 was measured.

Sequence data from this article can be found in the GenBank/EMBL data libraries under accession numbers EF144171, EF144173, and EF144174.

Supplemental Data

The following materials are available in the online version of this article.

Supplementary Material

[Supplemental Data]

Acknowledgments

We thank Dr. S.P. Dinesh-Kumar at Yale University for providing the TRV-silencing vectors.

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