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Jun 2, 2014 - The peptide elicitor PIP-1 (YGIHTH-nh2) induced various defense responses in tobacco cells. Types of defense responses induced by PIP-1 ...
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Continuous Stimulation of the Plant Immune System by the Peptide Elicitor PIP‑1 Is Required for Phytoalexin Biosynthesis in Tobacco Cells Yonghyun Kim, Eriko Komoda, Masahiro Miyashita,* and Hisashi Miyagawa Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Kyoto 606-8502, Japan S Supporting Information *

ABSTRACT: The peptide elicitor PIP-1 (YGIHTH-NH2) induced various defense responses in tobacco cells. Types of defense responses induced by PIP-1 were different based on its concentration range: oxidative burst (an early response) was induced at low micromolar levels, but phytoalexin production (a late response) required about 10−50-fold higher concentrations than those required for oxidative burst. We assumed that rapid decreases in the PIP-1 concentration due to enzymatic hydrolysis in the culture media could cause this difference. To examine the potential impact of such degradation particularly on induction of phytoalexin biosynthesis, we designed a degradation-resistant analogue, MePIP-1, in which the amide bond between the fifth and sixth residues was N-methylated. MePIP-1 was considerably more stable than PIP-1 and induced significant phytoalexin production upon treatment at low micromolar levels. Further investigation of the mechanism of action of MePIP-1 showed a requirement of continuous elicitor stimulation for 3−6 h for the phytoalexin production, which is likely to be regulated by longlasting MAP kinase activation. KEYWORDS: peptide elicitor, plant immunity, phytoalexin, MAP kinases

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INTRODUCTION Plants face challenges from a variety of pathogens such as bacteria, fungi, and viruses. To maintain their growth and subsequent reproduction against pathogens, plants have evolved unique immune systems.1,2 When plants sense pathogenic infections, they produce various defense responses, including oxidative burst, reinforcement of the cell wall, phytoalexin synthesis, and hypersensitive response (HR), to protect themselves.3−5 Plants recognize various molecules derived from the pathogens, which are referred to as either elicitors or pathogen-associated molecular patterns, to initiate a series of defense responses.6 Elicitors with various chemical properties have been reported. Peptides are among the most well-studied elicitors, which include the following: flg22, a 22 amino acid peptide from bacterial flagellin; elf18, an 18 amino acid peptide from bacterial elongation factor Tu; and Pep13, a 13 amino acid peptide from transglutaminase of Phytophthora sojae.7−9 In addition, several endogenous peptide elicitors released from host plants upon wounding and/or infection were identified. For example, a 23 amino acid peptide AtPep1 was isolated from Arabidopsis and its orthologue ZmPep1 from maize.10,11 These elicitors are mainly perceived by distinct receptor-like kinases (RLKs). RLKs activate the downstream signaling networks controlled by mitogen-activated protein (MAP) kinase and/or calcium-dependent protein kinase cascades, which further regulate the production and the activity of transcription factors, enzymes, hormones, and phytoalexins.12 Recently, we discovered a peptide elicitor (YGIHTH-NH2, PIP-1) by screening of a synthetic combinatorial random hexapeptide library.13 PIP-1 induces typical defense responses, such as oxidative burst, phytoalexin biosynthesis, and defense© 2014 American Chemical Society

related gene expressions, in tobacco cells via a jasmonic acid signaling pathway. Intriguingly, the types of defense responses induced by PIP-1 were different based on its concentration range: oxidative burst, which is observed immediately after treatment of PIP-1, was induced at low micromolar concentrations, but for the induction of capsidiol biosynthesis (Figure 1), which is induced 6−24 h after treatment, 10−50-

Figure 1. Structure of capsidiol.

fold higher concentrations than those required for oxidative burst were necessary. Since PIP-1 is a linear short peptide, it readily undergoes enzymatic degradation in and/or on the surface of the plant cells. Although the exact fate of applied PIP1 after treatment in the culture media of tobacco cells has not been examined yet, the decreases in PIP-1 concentrations due to degradation may affect the duration of the stimulation of immune systems, leading to failure to induce late-stage defense responses such as phytoalexin biosynthesis. In the present study, we hypothesized that inhibition of enzymatic degradation of PIP-1 can change the profile of Received: Revised: Accepted: Published: 5781

April 9, 2014 May 30, 2014 June 2, 2014 June 2, 2014 dx.doi.org/10.1021/jf501679p | J. Agric. Food Chem. 2014, 62, 5781−5788

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using a 150 mm × 2.1 mm i.d., 3 μm, Discovery BIO wide pore C18 column (Supelco, St. Louis, MO) for quantitation. The following condition was used for HPLC analysis: flow rate, 0.2 mL/min; column temperature, 40 °C; mobile phase, 0.1% TFA in H2O (solvent A) and 0.1% TFA in acetonitrile (solvent B); gradient, 10−30% solvent B for 20 min; UV detection, 215 nm. The amount of peptides in the medium was determined based on a calibration curve constructed using the synthesized standard peptides. Degradation products were identified by comparison of their m/z values and retention times on ESI-LC/MS analysis using an LCMS-2020 (Shimadzu) with those of the synthesized standard peptides. Measurement of H2O2 Generation. H2O2 generation was measured as described previously.15 Briefly, tobacco cells (0.2 g) resuspended in 1 mL of assay buffer (175 mM mannitol, 0.5 mM CaCl2, 0.5 mM K2SO4, 2.0 mM MES, pH 5.75) were incubated for 1 h at 25 °C using a tube rotator. After incubation, the cells were treated with PIP-1 analogues (10 μL) and incubated for a given time period (0−12 h) including 2 min to allow cell sedimentation. The supernatants (60 μL) of cell suspension were mixed with the H2O2 indicator solution (140 μL; 100 mM DA-64, 1 U/mL horse radish peroxidase, 100 mM PIPES, pH 7.0) in a 96-well microplate, and incubated for 10 min at 37 °C. The absorbance at 727 nm was read with a Benchmark microplate reader (Bio-Rad, Hercules, CA). The amount of H2O2 was determined based on a calibration curve. Quantitation of Capsidiol. Capsidiol was quantitated as described previously.13 Tobacco cells (0.6 g) resuspended in 3 mL of LS medium were incubated for 1 h. The cells were treated with PIP1 analogues and incubated for 24 h at 25 °C on a rotary shaker under continuous illumination. After incubation, the culture medium was extracted twice with 4 mL of ethyl acetate and dried in vacuo. The residue was dissolved in 1 mL of ethyl acetate and subjected to QP2010 gas chromatography/MS (GC/MS) analysis (Shimadzu), equipped with a 30 m × 0.25 mm, 0.25 μm phase thickness, DB5MS capillary column (J&W Scientific, Folsom, CA). For GC separation, helium was used as a carrier gas (100 kPa), and splitless injections were carried out at 220 °C. The column temperature was initially set at 100 °C for 2 min, then increased up to 270 °C at a rate of 4 °C/min for 42.5 min, and held at 270 °C for 2 min. The interface and ion source were set at 270 and 200 °C, respectively. Spectra were acquired in electron ionization mode with a scan range of m/z 45− 500. Capsidiol was identified by comparison of spectra and retention times with authentic standards obtained from Capsicum annuum fruits.16 The amount of capsidiol was quantitated from peak areas of the total ion chromatogram, based on a calibration curve constructed with authentic standards. Cell Death Assay. Cell death assay was performed as described previously with slight modifications.17 Tobacco cells (0.2 g) resuspended in 1 mL of LS medium were incubated for 1 h. The cells were treated with PIP-1 analogues and incubated for 24 h at 25 °C using a tube rotator. After incubation, the solution of Evans blue (Wako Pure Chemical Industries) was added into the cell suspension to a final concentration of 0.01%. The cells were reincubated for 15 min and then washed 3 times with distilled water to remove the unabsorbed dye. Washed cells were transferred into 50% methanol containing 1% SDS to extract the dye in the dead cells. After incubation for 1 h at 60 °C, the cells were centrifuged for 15 min at 1900g. Absorbance at 595 nm of the supernatant was read on a microplate reader (Bio-Rad). Analysis of Gene Expressions. Tobacco cells (0.6 g) resuspended in 3 mL of LS medium were incubated for 1 h at 25 °C. The cells were treated with PIP-1 analogues for a given time period (0−6 h). After removal of the medium by filtration, cells were ground in liquid nitrogen using a mortar and pestle. Total RNA was extracted with Trizol reagent (Life Technologies, Carlsbad, CA) according to the manufacturer’s instructions. The RNA (40 μg) was treated with 10 U of RQ1 RNase-free DNase (Promega, Madison, WI) for 30 min at 37 °C and then extracted by phenol and chloroform. Resultant RNA (1 μg) was reverse transcribed to cDNA in a final volume of 20 μL by using a PrimeScript RT reagent kit (Takara Bio, Otsu, Japan) according to the manufacturer’s instructions. For

induced defense responses in tobacco, in which oxidative burst and phytoalexin production would be induced at the similar concentration range. Therefore, a degradation-resistant PIP-1 analogue was designed based on the degradation profile of PIP1. Using this analogue, we examined the effect of elicitor stabilization on induction of defense responses including oxidative burst, phytoalexin biosynthesis, and HR-like cell death. To our knowledge, this is the first report demonstrating that enzymatic degradation of elicitors in the culture media considerably affects the induction of defense responses, particularly those in the late stage, such as phytoalexin biosynthesis in tobacco cells.

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MATERIALS AND METHODS

Plant Cell Culture. Suspension-cultured tobacco cells (Nicotiana tabacum cv. Xanthi) were grown in a 100 mL Erlenmeyer flask with 25 mL of liquid Linsmaier and Skoog (LS) medium at pH 5.75 containing 1-napthalenacetic acid (10 μM), 5-benzyladenine (1 μM), and 3% sucrose. The cell suspensions were maintained at 25 °C under continuous illumination on a rotary shaker (100 rpm) with subculture every 2 weeks and used at 9 and 10 days after subculture for experiments. Chemicals. N-(9-Fluorenylmethoxycarbonyl) (Fmoc)-protected amino acids, Fmoc-NH-SAL resin, and Cl-Trt(2-Cl) resin were purchased from Watanabe Chemical Industries (Hiroshima, Japan). (Carboxymethylaminocarbonyl)-4,4′-bis(dimethylamino)-diphenylamine sodium salt (DA-64) was obtained from Wako Pure Chemical Industries (Osaka, Japan). The peptides YGIHTH-NH2, YGIHTH-OH and D-(YGIHTH)-NH2 were previously synthesized in our laboratory.13 K252a was obtained from LC laboratories (Woburn, MA). Synthesis of Peptide Analogues. PIP-1 analogues (YGIHT-OH, YGIH-OH, and YGIHTmeA-NH2) were manually synthesized by using the Fmoc-based solid-phase peptide synthesis technique. The Cterminal amino acids of YGIHT-OH and YGIH-OH were attached to ClTrt(2-Cl) resin, and that of YGIHTmeA-NH2 was attached to FmocNH-SAL resin. Fmoc was deprotected with 20% piperidine in N,Ndimethylformamide (DMF) three times for 3, 3, and 20 min. Each Fmoc-protected amino acid (3 equiv) was coupled in the presence of O-(benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (3 equiv), 1-hydroxybenzotriazole (3 equiv), and N,Ndiisopropylethylamine (6 equiv) in DMF. The reaction was monitored by Kaiser test.14 After completion of the coupling reaction, the resin was sequentially washed with DMF, diethyl ether, and methanol and dried in vacuo. Cleavage of peptides from the resin and removal of side-chain protecting groups were carried out using the solution A (trifluoroacetic acid (TFA)/triisopropylsilane (TIPS)/dimethoxybenzene (92.5:2.5:5, v/v/v)) for YGIHTmeA-NH2 and solution B ((TFA/ TIPS/distilled water (95:2.5:2.5, v/v/v)) for YGIHT-OH and YGIHOH, which were reacted with each resin for 2 h at room temperature. After the resin was removed by filtration, cold diethyl ether was added to the filtrate to precipitate the peptides, followed by washing with cold diethyl ether twice and drying in vacuo. The desired peptides were purified by high-performance liquid chromatography (HPLC) using a 250 mm × 10 mm i.d., 5 μm, protein and peptide C18 semipreparative column (Grace, Columbia, MD), and lyophilized. Peptides were identified by electrospray ionization liquid chromatography/mass spectrometry (ESI-LC/MS) on a Shimadzu LCMS-2010 (Kyoto, Japan). Evaluation of Degradation of PIP-1 and Its Analogues. Tobacco cells were collected using Miracloth (Calbiochem, San Diego, CA), and 0.2 g of cells was resuspended in a microtube containing 1 mL of LS medium. The cells were incubated for 1 h at 25 °C by a tube rotator, and the solution of PIP-1 or its analogues in distilled water (final concentration of 10 μM) was added to the cells. After incubation at 25 °C for a given time period (0−24 h), the media were collected and filtered through syringe filters (0.45 μm pore size). The filtrates were frozen in liquid nitrogen and stored at −80 °C to prevent further peptide degradation. Each sample was subjected to HPLC analysis 5782

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previously.13 On the other hand, only a marginal level of capsidiol production (0.04 ± 0.07 μg/g FW) was observed upon treatment with 10 μM of PIP-1, which is a 4.5-fold higher concentration than that required for a half-maximum response of oxidative burst. Oxidative burst is an early stage defense response initiated within 5 min, whereas phytoalexin production is a late-stage response observed 6−24 h after elicitor treatment.23 Since linear peptides such as PIP-1 could be easily degraded in the presence of living cells, we assumed that a rapid decrease in PIP-1 concentrations due to the degradation was attributed to the requirement of high concentrations to induce phytoalexin biosynthesis. This also implies that early short stimulation by PIP-1 may not be enough for induction of phytoalexin biosynthesis. To explore this hypothesis, we first examined the effect of the duration of PIP-1 stimulation on capsidiol production by using various sample treatment procedures. When PIP-1 was removed from the culture media by washing the cells with fresh medium 1 h after treatment at 100 μM, no capsidiol production was detected. To further confirm that long-term stimulation by PIP1 is required for capsidiol production, a small amount of PIP-1 was repeatedly added (1 μM, 10 times) to the medium over 3 h, which amounted to only 10 μM at most. This repeated treatment induced a significant level of capsidiol (2.17 ± 0.61 μg/g FW), indicating that continuous stimulation of the immune system by PIP-1 is required for capsidiol production in tobacco cells. These results motivated us to synthesize a degradation-resistance PIP-1 analogue, which should allow the induction of phytoalexin biosynthesis at low micromolar concentrations. Design of Degradation-Resistant PIP-1. Prior to finding a way of preventing the degradation of PIP-1, we investigated its degradation process in the presence of tobacco cells by LC/ MS analysis. While PIP-1 (m/z observed for [M+2H]2+: 363.8) was detected in the extracellular medium at a retention time of 13.8 min immediately after its treatment at 10 μM, a new peak with m/z of 364.3 at a retention time of 14.9 min appeared as early as after 15 min (Figure 2). The observed molecular mass

semiquantitative reverse transcription polymerase chain reaction (RTPCR), 1 μL of the cDNA template was used for PCR in a solution (final volume of 20 μL) containing 0.5 U of TaKaRa Ex Taq (Takara Bio), 200 μM each of dNTP mixture, and 0.3 μM each of forward and reverse primers: SIPK, 5′-GATGTCTGATGCGGGGGCGG-3′ and 5′-GTGGCTCAACGTCGCCGGAA-3′; WIPK, 5′-CGGTGGAGGTCAATTCCCTG-3′ and 5′-CATTTACCAAAAGGTTGCTC3′; 18 Actin, 5′-CCATTGGCTCAGAGAGGTTC-3′ and 5′GTTGGAAGGTGCTGAGAG-3′.19 PCR was performed on a T100 thermal cycler (Bio-Rad) at 95 °C for 3 min (first cycle), 95 °C for 30 s (denaturation), 54−56 °C for 30 s (annealing), 72 °C for 60 s (extension; 26−28 cycles), and 72 °C for 5 min (last cycle). PCR products were separated on a 2% or 3% agarose gel and stained by EtBr (0.5 μg/L) for visualization. For quantitative RT-PCR, 2 μL of the cDNA template was amplified on a Thermal Cycler Dice (Takara Bio) in the presence of amplification buffer (25 μL of final volume) containing SYBR Premix Ex TaqII (Takara Bio) and 0.4 μM each of following forward and reverse primers: HMGR2, 5′-GAGGGGAGAGGAAAGTCTGTAG-3′ and 5′-TCTCTATGTTCTGAGCTGGGTC-3′; EAS, 5′-GTAAGGACTCATGCTGACGA-3′ and 5′-TCCACCACCTTGATACTTCG-3′. The relative quantities were determined using Actin as a reference gene. In-Gel Kinase Assay. Protein extraction and an in-gel kinase assay were performed as described previously with modifications.20 Tobacco cells (0.6 g) resuspended in 3 mL of LS medium were incubated for 1 h at 25 °C. Cells were treated with PIP-1 analogues for a given time period (0−9 h). After removal of medium, tobacco cells were ground in liquid nitrogen using a mortar and pestle. Powdered tobacco cells were homogenized with 1.5 mL of extraction buffer (50 mM HEPES, pH 7.4, 5 mM EDTA, 5 mM EGTA, 5 mM DTT, 10 mM NaF, 10 mM Na3VO4, 50 mM β-glycerophosphate, 1 mM 4-(2-aminoethyl)benzenesulfonyl fluoride, 2 μg/mL antipain, 2 μg/mL aprotinin, and 2 μg/mL leupeptin) and centrifuged at 16 000g for 30 min at 4 °C. Supernatants were quickly frozen in liquid nitrogen and stored at −80 °C. Extracts containing 10 μg of proteins were electrophoresed on 12% SDS-polyacrylamide gels embedded with 0.25 mg/mL myelin basic protein (MBP) (Sigma, St. Louis, MO) as a kinase substrate. After electrophoresis, the gel was washed with washing buffer (20% 2propanol, 50 mM Tris, pH 8.0) three times for 20 min each at room temperature to remove SDS and incubated with buffer A (50 mM Tris, pH 8.0, 5 mM DTT) for 1 h. The proteins in the gel were denatured in denaturation buffer (50 mM Tris−HCl, pH 8.0, 6 M guanidine·HCl, 20 mM DTT, 2 mM EDTA) twice for 30 min each. The kinases were allowed to renature on incubation with buffer A containing 0.04% Tween 20 overnight at 4 °C with five changes of buffer. After equilibration with assay buffer (50 mM HEPES, pH 8.0, 2.0 mM DTT, 0.1 mM EGTA, and 20 mM MgCl2) for 30 min at room temperature, the gel was incubated in 20 mL of assay buffer containing 30 μM ATP and 1.85 MBq of [γ-32P]-ATP (111 TBq/mmol) (PerkinElmer, Waltham, MA) for 1 h at room temperature. The reaction was stopped by transferring the gel into the solution containing 10% trichloroacetic acid (w/v) and 1% sodium phosphate (w/v). The gel was washed five times with the same solution for 2 h each to remove unincorporated [γ-32P]-ATP and dried on filter paper using a vacuum gel-dryer. The dried gel was exposed to BAS-MS2040 imaging plates (Fujifilm, Tokyo, Japan) and analyzed using BAS-2500 (Fujifilm). Prestained size markers (Nacalai Tesque, Kyoto, Japan) were used to estimate the size of the kinases.

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Figure 2. Degradation of PIP-1 (10 μM) in the culture medium of tobacco cells analyzed by LC/MS. Only LC/UV chromatograms were shown. Each peak was identified using synthesized standards.

RESULTS AND DISCUSSION Phytoalexin Production Induced by PIP-1. Capsidiol is a major sesquiterpene phytoalexin accumulated in the extracellular medium of tobacco cells after elicitor treatment (Figure 1), which is synthesized through the mevalonate pathway in plants.21,22 Treatment with PIP-1 at a 100 μM concentration induced a significant amount of capsidiol (4.24 ± 0.51 μg/g FW) in tobacco cells, whereas no capsidiol was detected after treatment with distilled water alone as reported

was consistent with that of C-terminally deamidated PIP-1 (YGIHTH-OH, m/z calculated for [M+2H]2+: 364.2).13 After 30 min, two other peaks were detected at the retention times of 15.2 min (m/z 245.3) and 17.4 min (m/z 295.7), which were respectively identified to be YGIH-OH (m/z calculated for [M +2H]2+: 245.1) and YGIHT-OH (m/z calculated for [M+2H]2+: 295.7), based on the comparison with synthesized standards. 5783

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Thereafter, PIP-1 and these degradation products underwent further degradation, and neither of them could be detected 3 h after the treatment. Thus, PIP-1 was found to be hydrolyzed rapidly from its C-terminal residue. This degradation could be catalyzed by proteases present in the extracellular space because PIP-1 cannot enter the cells due to its hydrophilic nature. In plants, several proteases have been identified from the extracellular space,24 some of which are induced by pathogen infection.25 Although further investigation is needed, proteases responsible for degradation of PIP-1 might be associated with defense responses in tobacco cells. The bioassay data showed that, while YGIHTH-OH was significantly elicitor-active, YGIHT-OH and YGIH-OH were practically inactive, so it is noted that the activity was lost in the media within 3 h at the longest. This result supports the findings that the repeated additions of PIP-1 could induce the capsidiol production even at low concentrations. On the basis of this degradation profile, we designed a degradation-resistant analogue of PIP-1 (Figure 3). Since

Figure 4. Comparison of degradation profiles of PIP-1 (10 μM) and MePIP-1 (10 μM) in the culture medium of tobacco cells. Vertical bars indicate ± SD of three independent replicates.

adsorption to tobacco cell surfaces rather than degradation. To confirm this, we evaluated the effect of adsorption using an elicitor-inactive but degradation-resistant PIP-1 analogue (DPIP-1), which is composed of all D-forms of amino acid residues.13 As expected, D-PIP-1 showed a decrease similar to MePIP-1 through 24 h incubation with tobacco cells. These results clearly indicate that MePIP-1 is highly resistant to enzymatic degradation in the presence of tobacco cells. Defense Responses Induced by MePIP-1. We investigated the effect of degradation-resistance of MePIP-1 on defense responses induced in tobacco cells. In terms of the oxidative burst, both PIP-1 and MePIP-1 induced similar H2O2 production profiles over 12 h (Figure 5A). On the other hand, a significant difference was observed for capsidiol production between PIP-1 and MePIP-1. Treatment with 10 μM MePIP-1 induced significant capsidiol production (4.7 μg/g FW), whereas marginal levels of capsidiol were induced by 10 μM PIP-1 (Figure 5B). To confirm that this difference was due to its degradation-resistance, MePIP-1 was removed from the medium by washing the cells after treatment. As expected, no capsidiol production was observed when MePIP-1 was removed 1 or 3 h after treatment (Figure 5C). However, washing the cells 6 or 9 h after the treatment had no effect on the induction of capsidiol production, although the amount was relatively low. These results indicate that the degradation-resistance of MePIP-1 critically contributes to its higher potency in inducing capsidiol production by enabling the continuous stimulation of the immune system. Induction of HR-like cell death, which is also a late-stage defense response, was then examined (Figure 5D). As expected, a significant difference was observed between MePIP-1 and PIP-1: only MePIP-1 induced cell death when they were treated at 10 μM. This suggests that continuous stimulation by PIP-1 is also required for the HR induction, like in the case of capsidiol production. Effects of MePIP-1 on MAP Kinase Activity. We then investigated the factors involved in triggering capsidiol biosynthesis in tobacco cells through continuous stimulation by MePIP-1. MAP kinase cascades play pivotal roles in signaling events associated with the recognition of pathogen and/or elicitors, which crucially affect downstream signaling events for induction of defense responses.12 It is known that activation of salicylic acid induced protein kinase (SIPK) and/ or wound-induced protein kinase (WIPK), representative MAP kinases in tobacco, is involved in defense responses induced by elicitors.29,30 To investigate the possible involvement of MAP kinase activation in capsidiol production induced by MePIP-1, we first examined the effect of a kinase inhibitor on capsidiol production. When K252a, a general protein kinase inhibitor,

Figure 3. Structure of degradation-resistant PIP-1 analogue (MePIP1).

methylation of amide nitrogen is known to improve stability against enzymatic degradation,26,27 we introduced an Nmethylated Ala residue into PIP-1 to substitute the C-terminal His residue, taking the observed hydrolysis of PIP-1 from the C-terminal end into consideration. As the previous study showed that the substitution of the C-terminal His residue with Ala had little effect on elicitor activity,13 the PIP-1 analogue having N-methylated Ala at the C-terminus (MePIP-1) exhibited rather higher activity (about 3.5-fold) than PIP-1, in terms of oxidative burst induction. This is possibly due to a conformation change of the peptide as a consequence of Nmethylation at the C-terminal residue, which may favorably interact with the receptor.27,28 Since PIP-1 is likely to be recognized by the membranous receptors of plant cells,13 the effect of a possible increase in intracellular uptake due to Nmethylation can be ruled out. We then monitored the degradation of MePIP-1 and compared it with that of PIP-1. When the tobacco cells were treated at 10 μM, MePIP-1 was present at 78% of the initial concentration after 3 h and was still detectable at 30% even after 24 h (Figure 4). This was in good contrast with the case of PIP-1, which was rapidly decreased to 3% of the initial concentration in 1 h upon the treatment with at 10 μM and completely disappeared after 3 h. The survival time of PIP-1 was extended to some extent when its initial concentration was raised to 100 μM, but it was decreased to an almost undetectable level as early as 6 h after the treatment. In this case, however, a significant level of elicitor-active YGIHTH-OH was present even after 6 h, which likely contributed to the induction of capsidiol production at least in part. Since no degradation products of MePIP-1 were detected 24 h after treatment, apparent reduction of MePIP-1 was likely due to its 5784

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Figure 5. Comparisons of defense responses induced by PIP-1 and MePIP-1 in tobacco cells. (A) Time-course of H2O2 generation. Inset shows the enlarged view for 2 h after treatment. (B) Dose dependence of capsidiol production 24 h after treatment. (C) Effect of cell washing on capsidiol production. Amounts of capsidiol were quantitated 24 h after elicitation. (D) Dose dependence of HR-like cell death 24 h after treatment. Asterisks indicate significant differences at P < 0.05 according to the Student’s paired t test. Vertical bars indicate ± SD of three independent replicates.

was added to the tobacco cells before addition of MePIP-1, production of capsidiol was inhibited in a dose-dependent manner (Figure 6). This suggested that activation of MAP

Figure 6. Inhibitory effect of K252a on capsidiol production induced by MePIP-1. Amounts of capsidiol were quantitated 24 h after treatment with MePIP-1. Capsidiol production without treatment with K252a was set at 100%. Vertical bars indicate ± SD of three independent replicates.

kinases, such as SIPK or WIPK, may be associated with capsidiol production. Thus, the effect of MePIP-1 on activation of SIPK and WIPK was investigated to examine its potential relevance to continuous elicitor stimulation prerequisite for capsidiol production. We first examined the expression of SIPK and WIPK transcripts. Consequently, no significant difference was observed between PIP-1 and MePIP-1 for induction of SIPK and WIPK transcripts (Figure 7A). Since it is known that the activity of these MAP kinases is regulated post-translation-

Figure 7. Effect of MePIP-1 on activation of MAP kinases. (A) Accumulation of transcripts of MAP kinases in response to PIP-1 and MePIP-1 at 10 μM. Actin was used as a control. (B) Activation of MAP kinases after treatment with PIP-1 or MePIP-1 at 10 μM. Activity was monitored by the in-gel kinase assay using MBP as a substrate.

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genes after treatment either with PIP-1 or MePIP-1 using quantitative RT-PCR. Since continuous activation by MePIP-1 for 3−6 h was shown to be required for capsidiol biosynthesis in our result, we analyzed the gene expression 4 h after the treatment with PIP-1 or MePIP-1. Consequently, expression of these genes was induced by both PIP-1 and MePIP-1, and no statistically significant difference was observed between them (Figure 8). To further examine the effect of the degradation-

ally by phosphorylation, not by an increase in transcript or protein levels,29,30 we then examined whether these MAP kinases are differently activated by PIP-1 and MePIP-1 by an ingel kinase assay. Upon treatment of either PIP-1 or MePIP-1, a significant activation of a 48 kD kinase, which corresponds to SIPK, was observed. However, a considerable difference between PIP-1 and MePIP-1 was observed for their activation of SIPK in the late phase (Figure 7B). SIPK was activated 1 h after the treatment with PIP-1 but rapidly decreased to a base level after 6 h. In contrast, MePIP-1 induced a long-lasting activation of SIPK even up to 9 h after the treatment. When MePIP-1 was removed from the medium 1 h after the treatment by washing of the cells, SIPK activity was decreased 6 h after the treatment, which was similar to that of PIP-1 treatment. These results clearly indicate that a prolonged SIPK activation is induced by a continuous stimulation by MePIP-1. In addition, when K252a (1 μM) was pretreated, activity of SIPK was significantly suppressed. In this case, activation was observed 9 h after treatment, but it could have been caused by inactivation of the inhibitor, probably by its degradation.31 These results strongly suggest that a prolonged SIPK activation is necessary for the activation of downstream signaling events that could be associated with capsidiol production. It is known that the strength of a defense response can be influenced by the magnitude and duration of MAPK activation. For instance, cryptogein, a proteinaceous elicitor derived from Phytophthora cryptogea, caused a prolonged activation of both SIPK and WIPK, which is necessary for induction of late-stage defense responses, such as HR-like cell death and long-lasting production of reactive oxygen species, in tobacco cells.32 When cryptogein was removed from the medium 0.5 h after treatment, no induction of HR-like cell death was observed, which coincided with rapid inactivation of both SIPK and WIPK. On the other hand, fungal cell wall elicitor, which induced only transient SIPK activation, failed to induce the latestage responses such as HR-like cell death.29 Although the reason why the fungal cell wall elicitor could not induce prolonged SIPK activation is unknown at present, it is possible that the enzymatic degradation of the elicitor by plant cells may be responsible for transient SIPK activation, which leads to failure to induce the late-stage responses, like in the case of PIP1. Thus, the duration of SIPK activation may determine the induction of late-stage defense responses, which could be regulated transcriptionally and/or post-translationally. Capsidiol Biosynthesis-Related Gene Expression Induced by MePIP-1. To examine the possible involvement of transcriptional regulation of capsidiol biosynthesis through prolonged SIPK activation, expression of capsidiol biosynthesisrelated genes was investigated. Capsidiol is synthesized via the mevalonate pathway in plants.21,22 In this pathway, 3-hydroxy3-methylglutaryl CoA reductase (HMGR) is considered to be a key enzyme,33 which catalyzes the rate-limiting step in the biosynthesis of all isoprenoid compounds, including capsidiol. The transcription level of this gene is known to be regulated by elicitor recognition.34,35 Farnesyl pyrophosphate (FPP) is synthesized from mevalonate in several steps and is located at a final branch point for sesquiterpene and sterol synthesis. FPP is converted to capsidiol in several steps including the reaction catalyzed by 5-epi-aristolochene synthase (EAS), which is demonstrated to be induced in response to elicitor treatment.21 To evaluate whether the degradation-resistance of MePIP-1 affects the transcription level of these capsidiol biosynthesisrelated genes, we examined the expression of HMGR2 and EAS

Figure 8. Relative transcription levels of capsidiol biosynthesis-related genes in response to PIP-1 and MePIP-1 at 10 μM. Values are the mean of three independent replicates. Vertical bars indicate ± SD.

resistance of MePIP-1 on the expression of HMGR2 and EAS transcripts, time-course analysis for up to 18 h was conducted for both PIP-1 and MePIP-1. Again, no significant difference in these expression profiles was observed between them. These results indicate that the early short stimulation by PIP-1 is sufficient for expression of HMGR2 and EAS genes but not enough for capsidiol biosynthesis. On the other hand, no induction of these genes was observed when K252a (1 μM) was pretreated (Figure 8), suggesting that the activation of SIPK in the early phase (