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MAPK Signaling Regulates Nitric Oxide and NADPH Oxidase-Dependent Oxidative Bursts in Nicotiana benthamiana^[W],[OA]

^a Laboratory of Defense in Plant–Pathogen Interactions, Graduate School of Bioagricultural Sciences, Nagoya University, Chikusa, Nagoya 464-8601, Japan
^b Plant Pathology Laboratory, Graduate School of Bioagricultural Sciences, Nagoya University, Nagoya 464-8601, Japan

¹ Address correspondence to hyoshiok{at}agr.nagoya-u.ac.jp.

	ABSTRACT

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION METHODS REFERENCES

 
Nitric oxide (NO) and reactive oxygen species (ROS) act as signals in innate immunity in plants. The radical burst is induced by INF1 elicitin, produced by the oomycete pathogen Phytophthora infestans. NO ASSOCIATED1 (NOA1) and NADPH oxidase participate in the radical burst. Here, we show that mitogen-activated protein kinase (MAPK) cascades MEK2-SIPK/NTF4 and MEK1-NTF6 participate in the regulation of the radical burst. NO generation was induced by conditional activation of SIPK/NTF4, but not by NTF6, in Nicotiana benthamiana leaves. INF1- and SIPK/NTF4-mediated NO bursts were compromised by the knockdown of NOA1. However, ROS generation was induced by either SIPK/NTF4 or NTF6. INF1- and MAPK-mediated ROS generation was eliminated by silencing Respiratory Burst Oxidase Homolog B (RBOHB), an inducible form of the NADPH oxidase. INF1-induced expression of RBOHB was compromised in SIPK/NTF4/NTF6-silenced leaves. These results indicated that INF1 regulates NOA1-mediated NO and RBOHB-dependent ROS generation through MAPK cascades. NOA1 silencing induced high susceptibility to Colletotrichum orbiculare but not to P. infestans; conversely, RBOHB silencing decreased resistance to P. infestans but not to C. orbiculare. These results indicate that the effects of the radical burst on the defense response appear to be diverse in plant–pathogen interactions.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION METHODS REFERENCES

 
Rapid production of nitric oxide (NO) and reactive oxygen species (ROS), called the NO burst and the oxidative burst, respectively, plays a role in diverse physiological processes, such as resistance to biotic and abiotic stress, hormonal signaling, and development (Doke, 1983; Kwak et al., 2003; Bright et al., 2006; Grun et al., 2006; Takeda et al., 2008). Recently, NO has attracted attention as the radical that participates in innate immunity in plants. NO induces phytoalexin accumulation (Noritake et al., 1996), activation of the mitogen-activated protein kinase (MAPK; Clarke et al., 2000), and the expression of defense genes, such as Phe ammonia-lyase (PAL) and pathogenesis-related proteins (Durner et al., 1998). In animals, NO is produced by NO synthase (NOS). The sources of NO synthesis in plants include reduction in nitrite by nitrate reductase (NR), oxidation of Arg to citrulline by NOS, and a nonenzymatic NO generation system (Bethke et al., 2004). Although evidence for Arg-dependent NO synthesis in plants has accumulated, no gene or protein that has a sequence similar to known mammalian-type NOS has been found in plants (Butt et al., 2003; Garcia-Mata and Lamattina, 2003). Guo et al. (2003) identified a NOS-like enzyme from Arabidopsis thaliana (At NOS1) with a sequence similar to a protein that has been implicated in NO synthesis in the snail Helix pomatia. The At NOS1 protein has no NOS activity (Zemojtel et al., 2006); therefore, At NOS1 was renamed At NOA1 for NO ASSOCIATED1 (Crawford et al., 2006). However, Arabidopsis mutant noa1 is still useful for its phenotype, which shows reduced levels of NO in plant growth, fertility, hormonal signaling, salt tolerance, and plant-pathogen responses (Guo et al., 2003; He et al., 2004; Zeidler et al., 2004; Zhao et al., 2007). Knocking out or down NOA1 expression provides a powerful tool to analyze NO function.

ROS are also important signaling molecules in plant immunity. In animals, ROS are synthesized by the phagocyte enzymatic complex of NADPH oxidase, which consists of two plasma membrane proteins, gp91^phox (phox for phagocyte oxidase) and p22^phox. Cytosolic regulatory proteins, p47^phox, p67^phox, p40^phox, and Rac2, translocate to the plasma membrane to form the active complex after stimulation (Lambeth, 2004). However, no homologs of the p22^phox, p67^phox, p47^phox, and p40^phox regulators of the phagocyte NADPH oxidase were found in the Arabidopsis genome. However, a homolog of one component, human Rac GTPase, has been isolated from rice (Oryza sativa), and the constitutively active mutant of Rac activates ROS production (Wong et al., 2007). Plant NADPH oxidases designated as Respiratory Burst Oxidase Homolog (RBOH) have been identified as genes related to mammalian gp91^phox in various plants (Groom et al., 1996; Keller et al., 1998; Torres et al., 1998; Yoshioka et al., 2001, 2003; Sagi et al., 2004) and carry an N-terminal extension comprising two EF-hand motifs, suggesting that Ca²⁺ regulates their activity. RBOHs are localized on the plasma membrane (Kobayashi et al., 2006). Arabidopsis rbohD rbohF double mutants show greatly decreased ROS production in response to infection with avirulent Pseudomonas syringae pv tomato DC3000 and Hyaloperonospora parasitica and in response to abscisic acid (Torres et al., 2002; Kwak et al., 2003). Analysis of the loss of function of Nt RBOHD in tobacco cells (Nicotiana tabacum) showed loss of ROS production by elicitor treatment (Simon-Plas et al., 2002). We also showed that silencing Nb RBOHA and Nb RBOHB in Nicotiana benthamiana plants leads to less ROS production and reduced resistance to infection by the potato pathogen Phytophthora infestans (Yoshioka et al., 2003). These reports suggest that RBOH is a key regulator of ROS production and has pleiotropic functions in plants.

The MAPK cascade is a major conserved signaling pathway used to transduce extracellular stimuli into intracellular responses in eukaryotes (MAPK Group, 2002; Nakagami et al., 2005). In the MAPK signal transduction cascade, a MAPK is activated by a MAPK kinase (MAPKK), which itself is activated by a MAPKK kinase (MAPKKK). Many studies have extensively characterized plant MAPKs, including tobacco wound-induced protein kinase (WIPK; Seo et al., 1999) and salicylic acid–induced protein kinase (SIPK; Zhang and Klessig, 1997) and their orthologs in other plant species (Lee et al., 2004; Katou et al., 2005; Pedley and Martin, 2005). WIPK and SIPK participate either in N gene–dependent resistance to tobacco mosaic virus (TMV; Zhang and Klessig, 1998; Jin et al., 2003) or in Cf9-dependent resistance to Cladosporium fulvum–derived elicitor Avr9 (Romeis et al., 1999) in a gene-for-gene specific way and in response to plant species–specific elicitors, such as elicitins (Zhang et al., 2000). MEK2, a tobacco MAPKK, is upstream of both WIPK and SIPK (Yang et al., 2001). Expression of Nt MEK2^DD, a constitutively active mutant of MEK2, induces hypersensitive response (HR)-like cell death, defense gene expression, and generation of ROS, all of which are preceded by activation of endogenous WIPK and SIPK (Yang et al., 2001; Ren et al., 2002). We also cloned the potato (Solanum tuberosum) ortholog of tobacco MEK2, St MEK2, which was formerly described as St MEK1 (Katou et al., 2003). Heterologous expression of St MEK2^DD in N. benthamiana induces WIPK and SIPK activities (Katou et al., 2003) and then an oxidative burst accompanied by increased RBOHB expression (Yoshioka et al., 2003). MAPKKK was identified as an upstream activator of MEK2 and SIPK in N. benthamiana (del Pozo et al., 2004). Silencing the MAPKKK gene eliminated HR-like cell death caused by interaction between the P. syringae avirulence gene AvrPto and its cognate resistant gene Pto (del Pozo et al., 2004). Interestingly, HR-like cell death induced by MAPKKK overexpression is reduced not only by silencing MEK2 or SIPK, but also by silencing MEK1 or NTF6 (del Pozo et al., 2004). NPK1-MEK1/NQK1-NTF6/NRK1 is a pivotal MAPK cascade in the regulation of cytokinesis (Soyano et al., 2003; Sasabe et al., 2006). Like WIPK or SIPK, silencing NPK1, MEK1, or NTF6 attenuates N- and Pto-mediated resistance against TMV (Jin et al., 2002; Liu et al., 2004) and P. syringae AvrPto, respectively (Ekengren et al., 2003). These studies indicated that MAPK cascades MEK2-WIPK/SIPK and NPK1-MEK1-NTF6 participate in disease resistance in plants. In addition, recent work has shown that tobacco NTF4, which shares 93.6% identity with SIPK, is also activated by MEK2, and ectopic expression of NTF4 induces HR-like cell death (Ren et al., 2006). However, the relationship between these MAPK cascades and the radical burst in response to pathogen signals is not clear.

In this study, we investigated the roles of MEK2-WIPK/SIPK/NTF4 and MEK1-NTF6 cascades in the regulation of NO and oxidative bursts in N. benthamiana. Gain-of-function and loss-of-function analyses showed that the MEK2-SIPK/NTF4 cascade controls the NOA1-mediated NO burst and that MEK2-SIPK/NTF4 and MEK1-NTF6 cascades regulate the NADPH oxidase-dependent oxidative burst. We also show that the NO burst and the oxidative burst have distinct effects on resistance to P. infestans and Colletotrichum orbiculare (syn. C. lagenarium) in N. benthamiana.

	RESULTS

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION METHODS REFERENCES

 
St MEK2^DD Activates MEK2-WIPK/SIPK Cascade, and Nb MEK1^DD Activates MEK1-NTF6 Cascade
To examine the function of MEK1-NTF6 cascade, we first isolated the tobacco MEK1 homolog from N. benthamiana using PCR with primers derived from a sequence of Nt MEK1 using an N. benthamiana cDNA library as a template. Analysis of the deduced amino acid sequence of the PCR product showed that the product of this gene shares >99% identity with Nt MEK1, so it was designated Nb MEK1 (N. benthamiana MEK1). In plants, MAPKKs are activated through phosphorylation of two Ser/Thr residues in a conserved S/TxxxxxS/T motif by MAPKKKs (MAPK Group, 2002). Replacement of two Ser/Thr residues with Glu or Asp results in constitutive activation of plant MAPKK (Yang et al., 2001; Ren et al., 2002). To examine the function of MEK1, we constructed Nb MEK1^DD, a constitutively active mutant of Nb MEK1, in which the conserved Ser-219 and Thr-225 were converted to Asp. Nb MEK1^DD was expressed under the control of the 35S promoter of Cauliflower mosaic virus using Agrobacterium tumefaciens infiltration of N. benthamiana leaves. Immunocomplex (IC) kinase assay using anti-NTF6 antibody showed no activation of NTF6 by Nb MEK1^DD, probably because the amount of endogenous NTF6 was not sufficient to detect the activity by the antibody. Therefore, NTF6-HA (hemagglutinin) was coexpressed with Nb MEK1^DD-HA or with inf1, an elicitin elicitor produced by P. infestans (Kamoun et al., 2003), by Agrobacterium infiltration in N. benthamiana leaves. As shown in Figure 1A , we confirmed that NTF6 is activated by Nb MEK1^DD and INF1.

View larger version (74K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 1. NO Burst Induced by the MEK2-WIPK/SIPK Cascade but Not by the MEK1-NTF6 Cascade. (A) Effects of the expression of Nb MEK1DD, St MEK2DD, and inf1 on the activation of NTF6, WIPK, and SIPK. Protein was extracted from intact leaves (Cont) or from leaves 24 h after the inoculation with A. tumefaciens carrying the indicated gene constructs. Kinase activities of the extracts were assayed by IC kinase assay with anti-NTF6 antibody and WIPK- or SIPK-specific antiserum using myelin basic protein (MBP) as a substrate. Immunoblot analyses were done using anti-HA antibody. Protein loads were monitored by Coomassie blue (CBB) staining of the bands corresponding to ribulose-1,5-bisphosphate carboxylase (Rubisco) large subunit. The asterisk indicates a nonspecific protein. (B) Effects of the expression of inf1, St MEK2DD, and Nb MEK1DD on NO burst. N. benthamiana leaves were pretreated with A. tumefaciens containing the indicated gene constructs and an Agrobacterium infiltration buffer as a control for 36 h and then were infiltrated with 12.5 µM DAF-2 DA agent to detect NO. Sodium nitroprusside (SNP; 500 µM) was infiltrated into leaves with DAF-2 DA. The infiltrated leaf areas were monitored 1 h later under fluorescence stereomicroscopy. Signal intensities were quantified by color histogram analysis. Data are means ± SE from three independent experiments. Bar = 1 mm. (C) Effects of silencing MEK1 and MEK2 on NO burst. After 3 weeks of inoculation with the gene silencing constructs TRV:MEK1, TRV:MEK2, and TRV as a control, silenced leaves inoculated with A. tumefaciens containing the indicated gene expression constructs for 36 h were analyzed as described in (B). Data are means ± SE from four experiments. Bar = 1 mm.

INF1 and St MEK2^DD Induce NOA1-Mediated NO Burst
We reported previously that a NO burst is induced by INF1 in tobacco cell suspension culture (Yamamoto et al., 2004). To investigate whether MAPK cascades regulate the NO burst, St MEK2^DD and Nb MEK1^DD were expressed using Agrobacterium infiltration in N. benthamiana leaves; inf1 and β-glucuronidase (GUS) were also expressed as positive and negative controls, respectively. The NO burst was detected by 4,5-diaminofluorescein diacetate (DAF-2DA)–mediated fluorescence (Figure 1B). Sodium nitroprusside, a NO generator, produced DAF-2DA–mediated fluorescence (Figure 1B), and the inducible fluorescence was eliminated by 2-4-carboxyphenyl-4,4,5,5-tetramethyl-imidazoline-1-oxyl-3-oxide (cPTIO), a NO scavenger (Figure 2), indicating that DAF-2DA detects NO. At 36 h after infiltration, the NO burst was markedly induced by the expression of St MEK2^DD, but not by Nb MEK1^DD (Figure 1B), suggesting that the NO burst is regulated by MEK2-WIPK/SIPK cascade. Time-course experiments showed that the DAF-2DA–mediated fluorescence was detected within 24 h after the infiltration and gradually increased until 36 h; thereafter, the leaf tissues showed a cell death phenotype. The timing of the NO burst was well matched with protein levels of St MEK2^DD (see Supplemental Figure 1 online). Therefore, the fluorescence intensities were measured at 36 h in the following experiments.

View larger version (59K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 2. Effects of VIGS of NOA1 and NOS or NR Inhibitors on NO Burst Induced by INF1 and St MEK2DD. NO production was estimated as described in Figure 1B. A. tumefaciens–inoculated leaves were infiltrated with cPTIO (500 µM), L-NAME (5 mM), or tungstate (100 µM) for 1 h before infiltration of DAF-2DA. Data are means ± SE from four experiments. Bar = 1 mm.

NOA1 participates indirectly in Arg-dependent NOS activity in plants (Guo et al., 2003; Zhao et al., 2007). To determine whether NOA1 contributes to the NO burst induced by INF1 and St MEK2^DD, we isolated the NOA1 homolog from N. benthamiana (Kato at el., 2008) and silenced Nb NOA1 using the TRV-VIGS system. The transcripts in the silenced plants decreased compared with control plants infected with TRV alone (see Supplemental Figure 3A online). Silenced leaves were inoculated with A. tumefaciens harboring inf1, St MEK2^DD or GUS as a control. The NO generation induced by INF1 and St MEK2^DD significantly decreased in NOA1-silenced plants compared with TRV control-inoculated plants (Figure 2 ), suggesting that NOA1 participates in the NO burst induced by INF1 and St MEK2^DD. Furthermore, we examined effects of some inhibitors on NO generation. L-NAME, the NOS inhibitor N^G-nitro-L-Arg-methyl ester, significantly reduced NO generation induced by INF1 and St MEK2^DD in TRV control-plants but not in NOA1-silenced plants (Figure 2). By contrast, D-NAME, an inactive isomer of L-NAME, had no effect on NO burst induced by INF1 (see Supplemental Figure 4 online). The results suggested that NOA1 takes part in NO burst induced by INF1 and St MEK2^DD and are in agreement with previous reports showing the involvement of NOA1 in Arg-dependent NOS activity (Guo et al., 2003; Zhao et al., 2007). Tungstate, an NR inhibitor, also significantly decreased NO generation in both TRV control- and NOA1-silenced plants (Figure 2), suggesting that NR is also involved in NO burst induced by INF1 and St MEK2^DD.

SIPK and NTF4 Are Required for NO Burst Induced by INF1 and St MEK2^DD
We evaluated whether the NO generation induced by INF1 and St MEK2^DD depends on WIPK, SIPK, or both. We silenced WIPK and SIPK in N. benthamiana and confirmed a marked reduction in WIPK and SIPK transcripts in the silenced plants compared with control plants infected with TRV alone (Figure 3A ). The NO generation induced by INF1 and St MEK2^DD was slightly reduced in WIPK-silenced plants compared with that in TRV control plants (Figure 3B). By contrast, VIGS of SIPK and WIPK/SIPK markedly reduced the NO generation to near the control level (Figure 3B). These results indicate the involvement of SIPK in the INF1-induced NO burst.

View larger version (57K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 3. SIPK and NTF4 Participate in NOA1-Mediated NO Burst. (A) N. benthamiana leaves were inoculated with TRV:WIPK (W), TRV:SIPK (S), TRV:WIPK/SIPK (W/S), and TRV as a control. Total RNA was isolated from leaves 24 h after inoculation with A. tumefaciens containing inf1 and was used for RT-PCR with specific primers for WIPK, SIPK, and NTF4. The specificity of the primer pairs to SIPK or NTF4 was confirmed using cDNAs (pBin-Nb SIPK and pBin-Nb NTF4) as templates. (B) NO was measured as described in Figure 1B. Data are means ± SE from 10 experiments. Bar = 1 mm. (C) IC kinase assay and immunoblot analysis were performed using anti-HA antibody as described in Figure 1A. (D) NO production was estimated as described in Figure 1B. Data are means ± SE from four experiments. Bar = 1 mm.

Silencing SIPK Compromises the Expression of RBOHB Induced by St MEK2^DD but Not by INF1
MAPK cascade participates in signaling to the oxidative burst (Yang et al., 2001; Ren et al., 2002). Our previous study showed that INF1 and St MEK2^DD induce an oxidative burst accompanied by expression of RBOHB, which is an inducible form of the NADPH oxidase of N. benthamiana (Yoshioka et al., 2003). To assess whether the expression of RBOHB induced by INF1 and St MEK2^DD depends on WIPK, SIPK, or both, we silenced the two MAPKs in N. benthamiana. Silenced leaves were inoculated with A. tumefaciens containing inf1 or St MEK2^DD (Figure 4 ). We confirmed a reduction in accumulation of WIPK and SIPK transcripts in the silenced plants using RNA gel blot hybridization. The expression of RBOHB induced by St MEK2^DD eliminated SIPK- and WIPK/SIPK-silenced plants (Figure 4), indicating that the MEK2-SIPK cascade regulates the expression of RBOHB. Unexpectedly, INF1-induced RBOHB expression was not compromised by the silencing of either WIPK or SIPK (Figure 4). These results suggest that INF1-induced expression of the RBOHB gene can be mediated by means of other signal pathways in addition to the MEK2-SIPK cascade.

View larger version (74K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 4. VIGS of SIPK Compromises the Expression of RBOHB Induced by St MEK2DD but Not by INF1. N. benthamiana leaves were inoculated for gene silencing with TRV:WIPK (W), TRV:SIPK (S), TRV:WIPK/SIPK (W/S), and TRV as a control. Total RNA was isolated from the silenced leaves infiltrated with A. tumefaciens containing inf1 (top panel) or St MEK2DD (bottom panel) at the indicated times (hours). Equal amounts of RNA (10 µg) were electrophoresed on a 1.2% agarose gel containing formaldehyde and were transferred to a membrane. Ethidium bromide staining of rRNA bands is shown as a loading control. Accumulation of transcripts for WIPK, SIPK, or RBOHB was confirmed by RNA gel blot hybridization.

View larger version (33K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 5. Nb MEK1DD Induces RBOHB-Dependent Oxidative Burst. (A) Effects of the expression of inf1, St MEK2DD, and Nb MEK1DD on oxidative burst. N. benthamiana leaves were inoculated with A. tumefaciens containing inf1, St MEK2DD, Nb MEK1DD, or GUS as a control. These different inoculation sites on the same leaf were infiltrated with 0.5 mM L-012 solution (a reagent to detect ROS) 24 h after the inoculation and were monitored using a CCD camera. White circles, whose diameters are 1.5 cm, indicate areas infiltrated with L-012. Chemiluminescence intensities were quantified by a program equipped with a photon image processor. Data are means ± SE from eight experiments. (B) Profile of transcript accumulation of RBOHB induced by INF1, St MEK2DD, and Nb MEK1DD. Total RNA was isolated from leaves infiltrated with A. tumefaciens carrying the indicated gene expression constructs or infiltration buffer for infiltration control at the indicated times. RBOHB transcript accumulation was monitored by RNA gel blot hybridization; ethidium bromide staining of rRNA is shown as a loading control. (C) VIGS of NTF6 compromised the expression of RBOHB induced by Nb MEK1DD. Total RNA was isolated from NTF6-silenced leaves infiltrated with A. tumefaciens containing Nb MEK1DD at the indicated times. Transcript accumulation was monitored by RNA gel blot hybridization; ethidium bromide staining of rRNA is shown as a loading control. (D) Effects of VIGS of RBOHA and RBOHB on oxidative burst induced by INF1, St MEK2DD, or Nb MEK1DD. Silenced leaves inoculated with A. tumefaciens for 24 h were analyzed as described in (A). Data are means ± SE from four experiments.

Silencing SIPK and NTF6 Compromises the RBOHB-Dependent Oxidative Burst Induced by INF1
Both MEK2-SIPK and MEK1-NTF6 cascades might participate in the RBOHB-dependent oxidative burst (Figure 5). However, the downstream signaling pathways from INF1 to the oxidative burst are not clear. To evaluate the roles of SIPK and NTF6 in the RBOHB-dependent oxidative burst induced by INF1, we silenced SIPK and NTF6 in N. benthamiana. The effects of VIGS were monitored by RT-PCR, and each target gene was fully silenced (see Supplemental Figure 3C online). The silenced leaves were inoculated with A. tumefaciens containing inf1. The expression of RBOHB was partially compromised in NTF6-silenced plants compared with TRV control-inoculated plants (Figure 6A ). By contrast, double silencing of SIPK and NTF6 showed pronounced reduction of accumulation of RBOHB transcripts by INF1 (Figure 6A). Similarly, VIGS of SIPK and NTF6 showed a more marked reduction in ROS generation than the reduction in ROS generation in plants silenced for SIPK or NTF6 (Figure 6B). We examined the possibility that NTF4 also participates in the ROS generation as shown in NO generation because VIGS of SIPK cosilenced NTF4 (Figure 3). The overexpression of SIPK and NTF4 by Agrobacterium infiltration activated RBOHB gene expression and the oxidative burst (Figures 6C and 6D). The SIPK- and NTF4-induced oxidative bursts were abolished by silencing RBOHB (Figure 6D). These data indicate that both the MEK2-SIPK/NTF4 cascade and MEK1-NTF6 cascade are responsible for the RBOHB-dependent oxidative burst induced by INF1.

View larger version (30K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 6. SIPK, NTF4, and NTF6 Participate in the Oxidative Burst and the Expression of RBOHB Induced by INF1. (A) N. benthamiana leaves were inoculated with the gene silencing constructs TRV:NTF6 (NTF6), TRV:SIPK/NTF6 (S/NTF6), or TRV alone as a control. Total RNA was isolated from silenced leaves infiltrated with A. tumefaciens containing inf1 at the indicated times. Accumulation of RBOHB mRNA was monitored by RNA gel blot hybridization; ethidium bromide staining of rRNA is shown as a loading control. (B) Effects of VIGS of SIPK (S), NTF6 (NTF6), or SIPK/NTF6 (S/NTF6) on the oxidative burst induced by INF1 or GUS as a control. Silenced leaves inoculated with A. tumefaciens for 24 h were analyzed as described in Figure 5A. Data are means ± SE from twelve experiments. (C) Transcript accumulation of RBOHB induced by St MEK2DD, SIPK, and NTF4. Total RNA was isolated from leaves 24 h after inoculation with A. tumefaciens carrying expression constructs for the indicated proteins and was used for RT-PCR with specific primers for RBOHB. (D) TRV control- and RBOHB-silenced leaves were inoculated with A. tumefaciens containing the indicated gene expression constructs. ROS were measured 24 h after the inoculation as described in Figure 5A. Data are means ± SE from four experiments.

View larger version (84K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 7. Effects of VIGS of NOA1 and RBOHB on Susceptibility to P. infestans and C. orbiculare. (A) Effects of silencing of NOA1, RBOHB, or NOA1/RBOHB (N/B) on the phenotype of the leaves. Silenced leaves were photographed 3 weeks after initiation of VIGS by TRV infection with no additional challenge. (B) Susceptibility to P. infestans in the silenced plants. Silenced leaves were inoculated with drops of a P. infestans zoospore suspension (2 x 105 zoospores/mL). Inoculated leaves were photographed 4 d after the inoculation. Red boxes: close-up shown in lower photographs. (C) Susceptibility to C. orbiculare in the silenced plants. Silenced leaves were inoculated with C. orbiculare conidial suspension (1 x 106 conidia/mL) using a vaporizer. Inoculated leaves were photographed 6 d after the inoculation. Red boxes: close-up shown in lower photographs. (D) Evaluation of disease symptom and biomass of P. infestans. Disease symptoms were evaluated by the following scoring system: disease symptom as percentage of whole leaf: 0, <20%; 1, 20 to 50%; 2, >50%. Data are means ± SE from seven experiments. To determine the biomass of P. infestans, real-time PCR was performed with P. infestans–specific primers using DNA isolated from inoculated leaves in (B). Data are means ± SE from four experiments. (E) Number and size of lesion spots counted 6 d after inoculation with C. orbiculare. Diameter of lesion spots was recorded using the following scoring system: 0, <1 mm; 1, 1 to 2 mm; 2, >2 mm. Data are means ± SE from eight experiments.

C. orbiculare conidia were sprayed on the silenced leaves. Small dark-colored spot lesions, which reflect susceptibility, not HR cell death, appeared on the leaves 6 d after the inoculation (Figures 7C and 7E). The number and size of disease spots on NOA1- or NOA1/RBOHB-silenced leaves increased approximately twofold compared with TRV control leaves. The number and size of spots on RBOHB-silenced leaves were similar to those of TRV control leaves. We also used 5'-terminal regions of the target genes for silencing and confirmed the phenocopy in these silenced plants (see Supplemental Figure 8C online). These results indicate that NOA1, but not RBOHB, appears to participate in basal defense to C. orbiculare.

	DISCUSSION

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION METHODS REFERENCES

 
In this study, we performed Agrobacterium-mediated expression assays and VIGS as gain-of-function and loss-of-function analyses, respectively, and found that the MEK2-SIPK/NTF4 cascade controls the NOA1-mediated NO burst, and MAPK cascades MEK2-SIPK/NTF4 and MEK1-NTF6 regulate the NADPH oxidase-dependent oxidative burst as summarized in Figure 8 . Knockdown of NOA1 and RBOHB showed that NO and oxidative bursts participate in defense responses to pathogens.

View larger version (27K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 8. Scheme of Proposed Model for MAPK Cascade Signaling Pathway Leading to NO and Oxidative Bursts. INF1 induces NO burst by means of MEK2-SIPK/NTF4 cascade and oxidative burst by means of MEK2-SIPK/NTF4 and (NPK1)-MEK1-NTF6 cascades. Based on loss-of-function and gain-of-function analyses, SIPK and NTF4 play important roles in NOA1-mediated NO burst and Nb RBOHB–dependent oxidative burst. NTF6 is also responsible for Nb RBOHB–dependent oxidative burst. The question mark indicates unidentified MAPKKK(s).

It has been shown that NR, a key enzyme of nitrogen assimilation, is another enzyme capable of producing NO in plants (Lamattina et al., 2003; Yamamoto et al., 2003). Here, we found that silencing NOA1 did not completely inhibit the NO burst induced by INF1 and St MEK2^DD (Figure 2). Furthermore, the NO burst was significantly suppressed by tungstate, a NR inhibitor, in both TRV control- and NOA1-silenced plants (Figure 2). These results suggest that another factor, NR, also participates in the NO burst induced by INF1 and St MEK2^DD in agreement with the previous finding that NR1 partially contributes to the INF1-induced NO burst in N. benthamiana (Yamamoto-Katou et al., 2006). However, the NOA1 silencing concomitant treatment with tungstate did not result in complete inhibition of the NO generation, suggesting that the nonenzymatic NO generation system also contributes to the NO burst, as reported by Bethke et al. (2004).

Defense-related RBOHs, which play a pivotal role in ROS production in response to pathogen signals, seem to be regulated at the transcriptional and posttranslational levels. We indicated previously that a calcium-dependent protein kinase (St CDPK5) activates potato RBOHs by direct phosphorylation of the N-terminal regions (Kobayashi et al., 2007). CDPK appears to regulate a rapid and transient accumulation of hydrogen peroxide (H₂O₂; phase I burst) and a massive oxidative burst at 6 to 9 h after elicitor treatment (phase II burst; Yoshioka et al., 2001; Kobayashi et al., 2007). Liu et al. (2007) reported that conditional gain-of-function by Nt MEK2^DD causes rapid shutdown of carbon fixation reaction in chloroplasts, which could lead to the generation of ROS in chloroplasts under illumination. They concluded that the chloroplast burst occurs earlier than the NADPH oxidase-dependent oxidative burst by MAPK (phase II burst) and that the chloroplast-generated ROS are only a facilitator that accelerates cell death because plants cells without mature chloroplasts die eventually. They suggested that mitochondria-generated ROS might be essential in accelerating the cell death process. Communication between chloroplasts and mitochondria exists in cells undergoing HR cell death (Yao et al., 2004). Mitochondrial dysfunction can be caused by MEK2^DD. MEK2^DD-mediated dysfunction can be prevented by Bcl-xL, which is a mammalian anti-apoptotic factor that can prevent mitochondrial dysfunction in plants, the same as in animals (Takabatake et al., 2007). We routinely use the chemiluminescence probe L-012 to detect RBOH-generated ROS in N. benthamiana leaves. The probe has been used for the analysis of ROS generated by membrane-bound NADPH oxidase in neutrophils (Imada et al., 1999). In this study, we failed to detect rapid chloroplast- and mitochondria-generated ROS in elicited plants after treatment with INF1, St MEK2^DD, Nb MEK1^DD (Figure 5D), and fungal infection (see Supplemental Figure 7 online), suggesting that our method is suitable to detect apoplastic ROS generated by membrane-bound RBOH in plant cells.

We propose that early chloroplast-generated ROS caused by Nt MEK2^DD and phase I burst by elicitors might contribute to the influx of Ca²⁺ into cytoplasm and that the increased level of Ca²⁺ might result in activation of CDPK as an inducer of the phase II burst from NADPH oxidases localized in the plasma membrane.

Crosstalk between Two MAPK Cascades and Radical Bursts
The expression and activation of the Arabidopsis Ser/Thr kinase OXIDATIVE SIGNAL-INDUCIBLE1 (OXI1) are induced in response to H₂O₂ (Rentel et al., 2004). OXI1 is required for activation of MAPKs (MPK3 and MPK6, orthologs of WIPK and SIPK, respectively, in Arabidopsis) after treatment with H₂O₂ or an elicitor. In this study, we showed that in N. benthamiana the MEK2-SIPK cascade regulates the oxidative burst resulting from the induction of RBOHB expression (Figures 4 to 6). Together, we can assume the existence of a positive feedback circuit between SIPK and RBOHB. However, little is known about the crosstalk between MAPK cascades and NO production. In this study, we showed that the MAPK cascade MEK2-SIPK regulates the NO burst. NO also activates potential MAPKs, as shown by an in-gel kinase assay using MBP as a substrate (Clarke et al., 2000). SIPK may give a positive feedback between NO burst signals as well as oxidative burst signals.

A novel MAPKK (MKK1) has been identified as an effective inducer of HR-like cell death in N. benthamiana. MKK1 activates SIPK, and MKK1-mediated cell death is compromised by silencing SIPK (Takahashi et al., 2007). The transient expression of SIPK is sufficient to induce HR-like cell death and the expression of 3-hydroxy-3-methylglutaryl CoA reductase, a gene encoding a key enzyme in the phytoalexin biosynthesis pathway (Zhang and Liu, 2001). This study showed that SIPK regulates the expression of RBOHB and the NO burst. These results strongly indicate that SIPK plays a central role in multiple defense responses. However, our results also showed that silencing WIPK slightly compromises the NO burst induced by INF1 and St MEK2^DD (Figure 3B). WIPK could function in accelerating SIPK-mediated cell death or in initiating a new pathway (Liu et al., 2003). WIPK might function as a factor to commit SIPK-mediated NO. NTF4 is a tobacco MAPK that reveals high sequence similarity to SIPK (93.6% identity) and a similar expression pattern and activation profile of SIPK (Ren et al., 2006). The conditional expression of NTF4 induces autophosphorylation and HR-like cell death (Ren et al., 2006). In this study, we demonstrated that NTF4 also regulates the radical bursts similarly to SIPK. NTF4 may play a similar role to SIPK in the signaling of basal defense.

The NPK1-MEK1-NTF6 cascade, a pivotal MAPK cascade in the regulation of cytokinesis (Soyano et al., 2003), participates in N- and Pto-mediated resistance to TMV and P. syringae AvrPto, respectively (Ekengren et al., 2003; Jin et al., 2003; Liu et al., 2004). In this study, we showed that the MEK1-NTF6 cascade regulates the oxidative burst accompanied by induction of RBOHB expression (Figure 5). MEK1 has been shown to be an activator of NTF6 in cytokinesis (Soyano et al., 2003), and in this study, the NTF6 activation profile by Nb MEK1^DD was obtained only when NTF6 was overexpressed in N. benthamiana leaves (Figure 1), suggesting that the amount of endogenous NTF6 was not sufficient to detect the activity by anti-NTF6 antibody. However, we cannot rule out the possibility that another efficient MAPKK activating NTF6 might exist for INF1-induced oxidative burst. How NTF6 can bifurcate two distinct cellular functions, cytokinesis and innate immunity, is unclear. These puzzles require further investigations.

NOA1 and RBOH Play Distinct Roles in Resistance to P. infestans and C. orbiculare
The Arabidopsis noa1 knockout mutant shows reduction of NO production and basal defense in response to lipopolysaccharide and an increase in the susceptibility to virulent P. syringae (Zeidler et al., 2004), indicating that NOA1 participates in NO production and defense responses. In our previous study, VIGS of RBOHA and RBOHB in N. benthamiana reduced the oxidative burst and disease resistance to P. infestans (Yoshioka et al., 2003). These studies show that NOA1 and RBOH play pivotal roles in defense responses during plant–pathogen interactions.

Plant defenses are often initiated by a gene-for gene interaction between a dominant plant resistance gene and a pathogen avirulence gene, which provides race-specific resistance. Plants also use a much less specific recognition system that identifies pathogen-associated molecular patterns, such as INF1 (Kamoun et al., 1998) and cell wall elicitor of the potato pathogen P. infestans (Katou et al., 2005). Silencing inf1 in P. infestans enhances the susceptibility of N. benthamiana (Kamoun et al., 1998), suggesting that defense responses of N. benthamiana to P. infestans are based on the basal defense by recognition of pathogen-associated molecular patterns. The same is true for C. orbiculare and N. benthamiana interactions (Takano et al., 2006). Here, we showed that NOA1 and RBOHB play distinct roles in the resistance to P. infestans and C. orbiculare. NOA1 silencing induced high susceptibility to C. orbiculare, but not to P. infestans, whereas RBOHB silencing showed a strong effect on resistance to P. infestans, but not to C. orbiculare (Figure 7). These results indicate that the effects of the NOA1-mediated NO burst and the NADPH oxidase-dependent oxidative burst on defense response appear to be diverse in plant–pathogen interactions.

The Arabidopsis rbohD rbohF double mutant displays a marked reduction in ROS in response to infection with avirulent H. parasitica (Torres et al., 2002). However, Torres et al. (2002) found that the double mutant is not compromised in basal defense against a virulent isolate of H. parasitica, an oomycete like P. infestans. This contrast with our results might be due to the diversified effects of ROS on disease resistance dependent on each host–parasite interaction. Phytophthora species, such as Phytophthora brassicae (Roetschi et al., 2001) and several isolates of Phytophthora cinnamomi (Robinson and Cahill, 2003), can infect Arabidopsis. On the other hand, P. infestans cannot infect Arabidopsis (Huitema et al., 2003) and instead induces HR cell death accompanied by an oxidative burst in the attacked cell (Kobae et al., 2006). Transgenic potato plants expressing a H₂O₂-generating glucose oxidase are resistant to P. infestans (Wu et al., 1995). This result supports the idea that ROS play an important role in disease resistance to P. infestans. We believe that each pathogen has diverse strategies for infection. For instance, recent work has shown that Ustilago maydis, a biotrophic pathogen of maize (Zea mays), uses a redox sensor protein that controls the H₂O₂ detoxification system for coping with early plant defense responses (Molina and Kahmann, 2007). In the barley (Hordeum vulgare)–Blumeria graminis interaction, silencing Hv RBOHA, a barley ortholog of Arabidopsis RBOHF, during the penetration process of B. graminis leads to increased penetration resistance (Trujillo et al., 2006), which could be due to a decrease in host cell wall softening usually induced by plant RBOH-mediated superoxide (O₂^–).

ROS and NO are believed to play important roles independently or coordinately in plant innate immunity. ROS generated on the plasma membrane are released to the apoplast, inducing oxidative crosslinking of glycoproteins, strengthening the cell wall against secondary infection (Bradley et al., 1992) and simultaneously activating the Ca²⁺ channel to increase the level of cytosolic Ca²⁺ (Lecourieux et al., 2002). Ca²⁺ may function not only as an inducer of the oxidative burst but also as a signaling molecule downstream of the oxidative burst that causes various cellular responses, including defense (Torres and Dangl, 2005). However, NO signaling includes various messenger molecules, such as cGMP, cADP ribose, and Ca²⁺ (Durner et al., 1998; Wendehenne et al., 2001; Lamotte et al., 2004; Romero-Puertas et al., 2004), which both directly and indirectly modulate the expression of specific genes (Polverari et al., 2003; Parani et al., 2004). NO signaling pathways often include posttranslational modification of target proteins, such as NO-dependent Cys S-nitrosylation that can modulate the activity and function of different proteins (Sokolovski and Blatt, 2004; Feechan et al., 2005; Lindermayr et al., 2005; Romero-Puertas et al., 2007). NO can also react with O₂^– to form the reactive molecule peroxynitrite (ONOO^–). ONOO^– can lead to formation of NO₂ and the hydroxyl radical (OH^•). OH^• is a very strong oxidizing species that can rapidly attack biological membranes and all types of biomolecules, such as DNA and proteins, leading to irreparable damage, metabolic dysfunction, and cell death (del Rio et al., 2003). ONOO^– is also responsible for Tyr nitration (Lamattina et al., 2003), which is the major toxic reactive nitrogen species in animal cells (Stamler et al., 1992). We reported previously that treatment of BY-2 tobacco cells with INF1 induces ONOO^– generation, and we investigated Tyr nitration as a direct reaction of ONOO^– (Saito et al., 2006); that study supported the idea that NO and O₂^– are produced simultaneously through a convergent signaling pathway, that is, MAPK cascades in plants. ONOO^– is relevant to HR and defense gene expression (Alamillo and García-Olmedo, 2001). One study emphasized that the combination of NO and H₂O₂, but not ONOO^–, takes part in the induction of defense responses (Delledonne et al., 2001). NO or ROS are not essential for HR in plants but induce apoptosis in adjacent cells during the defense response (Tada et al., 2004). HR cell death may require a fine balance between NO and ROS (Delledonne et al., 2001). In this study, we showed that double silencing of RBOHB and NOA1 does not confer a synergistic effect on resistance to P. infestans and C. orbiculare compared with individual silencing of these genes (Figure 7; see Supplemental Figure 8 online), suggesting that ONOO^– might not be required for full defense responses to at least these two pathogens.

In regulating gene expression, NO can induce the expression of PAL and chalcone synthase independently of ROS, and induction by NO of defense-related genes, such as glutathione S-transferase, depends on H₂O₂ (Grun et al., 2006). In abscisic acid signal transduction for stomatal closure, NO and H₂O₂ induced by abscisic acid individually inhibit stomatal opening specific to blue light (Bright et al., 2006; Zhang et al., 2007). However, the cooperative and individual roles of NO and ROS during disease resistance are unclear. Transgenic potato plants expressing St MEK2^DD fused to a pathogen-inducible promoter are resistant to both the biotrophic pathogen P. infestans and the necrotrophic pathogen Alternaria solani, which causes early blight of Solanaceae plants, such as potato and tomato (Solanum lycopersicum) (Yamamizo et al., 2006). However, our preliminary data indicate that transgenic potato plants carrying St CDPK5VK driven by the same pathogen-inducible promoter show high resistance to P. infestans but high susceptibility to A. solani. St CDPK5VK induces only ROS production (Kobayashi et al., 2007), and in this study, we showed that St MEK2^DD induces both NO and ROS. These results support the results of this study that silencing NOA1 or RBOHB distinctly affects susceptibility to P. infestans and C. orbiculare. Plants may have obtained during evolution the signaling pathway regulating both NO and ROS production to adapt to a wide spectrum of pathogens.

	METHODS

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION METHODS REFERENCES

 
Plant Growth Conditions
Nicotiana benthamiana plants were grown at 25°C and 70% humidity under a 16-h photoperiod and an 8-h-dark period in environmentally controlled growth cabinets.

cDNA Cloning of Nb MEK1 and Site-Directed Mutagenesis
The cDNA of Nb MEK1 was amplified using PCR from an N. benthamiana cDNA library as a template (Yoshioka et al., 2003) using the following primers corresponding to the first 30 nucleotide sequences and 3'-terminal 30-nucleotide sequences of Nt MEK1, respectively: forward 5'-ATGAAGACGACGAAGCCATTGAAGGAACTT-3' and reverse 5'-TTATCTTGGAAAATTTACAGGAGGTTCCAG-3'. The PCR products were cloned into the TOPO plasmid vector (TOYOBO) and were sequenced. The constitutively active mutant of Nb MEK1, Nb MEK1^DD, with the conserved Ser-219 and Thr-225 replaced by Asp, was generated using a Mutan-Super Express Km kit (Takara). The sequences of the PCR products and Nb MEK1^DD were verified by sequencing with an ABI Prism BigDye Terminator Cycle Sequence kit (Perkin-Elmer).

Agrobacterium tumefaciens–Mediated Transient Expression in N. benthamiana
The cDNA fragment of constitutively active mutant Nb MEK1^DD was cloned into the pGreen binary vector (Hellens et al., 2000), in which a HA-tag was added to the C-terminal end. Nb MEK1^DD, St MEK2^DD (Katou et al., 2003), and GUS, which was used as a control for the effect of Agrobacterium infiltration, were preceded by the 35S promoter of Cauliflower mosaic virus, a 5'-untranslated region was replaced with the sequence from the TMV, and the nopaline synthase terminator region was on the 3'-end of the gene (Jones et al., 1992).

Binary plasmids were transformed into Agrobacterium strain GV3101, which included the transformation helper plasmid pSoup (Hellens et al., 2000) by electroporation. The overnight culture was diluted 10-fold in LB/kanamycin/rifampicin/tetracycline and was cultured to OD₆₀₀ 0.6. The cells were harvested by centrifugation and were resuspended in 10 mM MES-NaOH, pH 5.6, and 10 mM MgCl₂. The suspensions were adjusted to OD₆₀₀ 0.5, and acetosyringone was added to 150 µM. The bacterial suspensions were incubated for 2 to 3 h at 22°C and then were infiltrated into leaves of 4- to 5-week-old N. benthamiana plants using a needleless syringe (Yoshioka et al., 2003). A. tumefaciens carrying inf1 was prepared as described by Kamoun et al. (2003).

VIGS
VIGS was done as described by Ratcliff et al. (2001). The following primers were used to amplify cDNA fragments from N. benthamiana using the N. benthamiana cDNA library as a template (Yoshioka et al., 2003). Restriction sites were added to the 5'-end of forward or reverse primer for cloning into a TRV vector pTV00 (RNA2): Nb MEK1-F-AccI (5'-CCGGTCGACCTCAGAAACTAAGGAGATAGATCTT-3') and Nb MEK1-R-ClaI (5'-CCATCGATAAAACCTGCTTGCAAACAACTG-3') (restriction sites are underlined), which produced a 363-bp fragment; Nb MEK2-F-ClaI (5'-CCATCGATAGATGTGCCGTGAGATCGA-3') and Nb MEK2-R-ClaI (5'-CCATCGATGCCTCGAGTTGATTAGTAAATTCG-3'), which produced a 261-bp fragment; Nb NOA1-F-BamHI (5'-CGGGATCCCGGAGTCACAGATAGCTGACCG-3') and Nb NOA1-R-ClaI (5'-CCATCGATGGCTGTCCTCCAGCTTTGC-3'), which produced a 478-bp fragment; Nb WIPK-F-BamHI (5'-CGGGATCCCAGATGGTACAGGGCACCAG-3') and Nb WIPK-R-HindIII (5'-CCCAAGCTTCGACAAGATCAATAGCCAATGGG-3'), which produced a 302-bp fragment; Nb SIPK-F-ClaI (5'-CCATCGATTATGACGGAATATGTTGTGACAA-3') and Nb SIPK-R-ClaI (5'-CCATCGATTCTCGACAAGATCAATTGCA-3'), which produced a 323-bp fragment; Nb RBOHA-F-HindIII (5'-CCCAAGCTTCTGTGATTAACATTGACGCTC-3') and Nb RBOHA-R-BamHI (5'-CGGGATCCGCATTGTGCGAAATCGGAAC-3'), which produced a 500-bp fragment; Nb RBOHB-F-ClaI (5'-CCATCGATGTTAAACAAACGAGGCGGCA-3') and Nb RBOHB-R-HindIII (5'-CCCAAGCTTTACATTCTCCAAATTTGGCAC-3'), which produced a 500-bp fragment; and Nb NTF6-F-AccI (5'-CCGGTCGACCGCTAGAACCACTTCAGAG-3') and Nb NTF6-R-ClaI (5'-CCATCGATTAGAGCCTCGTTCCATATGAGC-3'), which produced a 535-bp fragment. N. benthamiana was infected by viruses by Agrobacterium-mediated transient expression of infectious constructs. pBINTRA6 (RNA1) and the pTV00 containing the inserts (RNA2) were transformed by electroporation separately into A. tumefaciens strain GV3101, which includes the transformation helper plasmid pSoup (Hellens et al., 2000). A mixture of equal parts of Agrobacterium suspensions of RNA1 and RNA2 was inoculated into 2- to 3-week-old N. benthamiana seedlings. The inoculated plants were grown under a 16-h photoperiod and an 8-h dark period at 23°C. The upper leaves of the inoculated plants were used for assays 3 to 4 weeks after inoculation.

Immunoblotting
For immunoblotting, the protein extracts (20 µg) were separated on a 12% SDS-polyacrylamide gel and were transferred to a nitrocellulose membrane (Schleicher and Schuell). After blocking in TBS-T (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, and 0.05% Tween 20) with 5% nonfat dry milk overnight at 4°C, the membranes were incubated with monoclonal anti-HA antibody (clone HA-7; Sigma-Aldrich) diluted with TBS-T at room temperature for 1 h. After washing with TBS-T, the membranes were incubated with horseradish peroxidase–conjugated anti-mouse Ig antibody (Amersham) diluted with TBS-T for 1 h at room temperature. The antibody-antigen complex was detected using the ECL Western-Blotting detection kit (Amersham) and the Light-Capture system (ATTO) and then quantified using the CS analyzer program (ATTO).

IC Kinase Assays
IC kinase assay was done as described by Zhang and Liu (2001). In the case of using anti-NTF6 antibody, the protein extracts (400 µg) were incubated with anti-NTF6 antibody (Soyano et al., 2003) in an immunoprecipitation buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1 mM EDTA, 50 mM NaF, 10 mM β-glycerophosphate, 1 mM Na₃VO₄, and 1% Triton X-100) at 30°C for 2 h with shaking. In the case of WIPK- or SIPK-specific antiserum, the protein extracts (50 µg) were incubated with the specific antisera in the immunoprecipitation buffer at 4°C for 2 h with shaking. Then each mixture was incubated with 40-µL aliquot of 50% (v/v) protein A-Sepharose (GE Healthcare) at 4°C overnight with shaking. In the case of using anti-HA agarose conjugate (Sigma-Aldrich), the protein extracts (200 µg) were incubated with anti-HA agarose conjugate in the immunoprecipitation buffer at 4°C overnight with shaking. Protein A-Sepharose complexes and anti-HA agarose conjugate were pelleted by brief centrifugation, washed three times with 1 mL of the immunoprecipitation buffer, and then were washed three times with 1 mL of kinase reaction buffer (20 mM HEPES-KOH, pH 7.6, 10 mM MgCl₂, and 1 mM DTT). The kinase activity in the complex was assayed at 25°C for 30 min in a final volume of 15 µL containing 0.4 mg/mL MBP, 100 µM ATP, and 8 nCi/mL of [-³²P]ATP. The reaction was stopped by adding SDS-PAGE sample loading buffer. After electrophoresis on a 15% SDS-polyacrylamide gel, the phosphorylated MBP was visualized by autoradiography.

NO Measurements
NO accumulation was monitored using NO-sensitive dye DAF-2DA (Daiichi Pure Chemicals). Leaves were infiltrated with 200 mM sodium phosphate buffer at pH 7.4, including 12.5 µM DAF-2DA using a needleless syringe and were incubated for 1 h in the dark at room temperature before observation. Fluorescence from diaminotriazolofluorescein (DAF-2T), the reaction product of DAF-2DA with NO, was observed using a fluorescent stereomicroscope (MZ FLIII; Leica) equipped with a CCD camera. The excitation was at 470 nm, and the emission images at 525 nm were obtained at constant acquisition time. The fluorescence intensity of the digital image was determined by color histogram analysis of Photoshop version 7.0 (Adobe).

ROS Measurements
ROS measurements were done as described by Kobayashi et al. (2007). The relative intensity of ROS generation was determined by counting photons from L-012–mediated chemiluminescence. The 0.5 mM L-012 in 10 mM MOPS-KOH at pH 7.4 was infiltrated into N. benthamiana leaves using a needleless syringe. Chemiluminescence was monitored continuously using a photon image processor equipped with a sensitive CCD camera (Aquacosmos 2.5; Hamamatsu Photonics) and quantified using the U7501 program (http://jp.hamamatsu.com/en/index).

RT-PCR
Total RNA from N. benthamiana leaves was prepared using TRIzol reagent according to the procedure of the manufacturer (Invitrogen). RT-PCR was conducted using a commercial kit (ReverTra Ace --; TOYOBO). PCR was performed by ExTaq (Takara) with denaturing, anneal