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Originally published In Press as doi:10.1074/jbc.M703344200 on September 14, 2007

J. Biol. Chem., Vol. 282, Issue 47, 34013-34018, November 23, 2007
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Identification of a Biosynthetic Gene Cluster in Rice for Momilactones*

Kazuhiro Shimura{ddagger}, Atsushi Okada{ddagger}, Kazunori Okada{ddagger}, Yusuke Jikumaru{ddagger}, Kwang-Wook Ko{ddagger}, Tomonobu Toyomasu§, Takeshi Sassa§, Morifumi Hasegawa, Osamu Kodama, Naoto Shibuya||, Jinichiro Koga**, Hideaki Nojiri{ddagger}, and Hisakazu Yamane{ddagger}1

From the {ddagger}Biotechnology Research Center, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan, the §Department of Bioresource Engineering, Yamagata University, Tsuruoka, Yamagata 997-8555, Japan, the College of Agriculture, Ibaraki University, 3-21-1 Chuo, Ami, Ibaraki 300-0393, Japan, the ||Department of Life Science, Meiji University, Kawasaki, Kanagawa, 214-8571 Japan, and the **Food and Health Research and Development Laboratories, Meiji Seika Kaisha, Ltd., Sakado, Saitama 350-0289, Japan

Received for publication, April 23, 2007 , and in revised form, September 14, 2007.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Rice diterpenoid phytoalexins such as momilactones and phytocassanes are produced in suspension-cultured rice cells treated with a chitin oligosaccharide elicitor and in rice leaves irradiated with UV light. The common substrate geranylgeranyl diphosphate is converted into diterpene hydrocarbon precursors via a two-step sequential cyclization and then into the bioactive phytoalexins via several oxidation steps. It has been suggested that microsomal cytochrome P-450 monooxygenases (P-450s) are involved in the downstream oxidation of the diterpene hydrocarbons leading to the phytoalexins and that a dehydrogenase is involved in momilactone biosynthesis. However, none of the enzymes involved in the downstream oxidation of the diterpene hydrocarbons have been identified. In this study, we found that a putative dehydrogenase gene (AK103462) and two functionally unknown P-450 genes (CYP99A2 and CYP99A3) form a chitin oligosaccharide elicitor- and UV-inducible gene cluster, together with OsKS4 and OsCyc1, the diterpene cyclase genes involved in momilactone biosynthesis. Functional analysis by heterologous expression in Escherichia coli followed by enzyme assays demonstrated that the AK103462 protein catalyzes the conversion of 3beta-hydroxy-9betaH-pimara-7,15-dien-19,6beta-olide into momilactone A. The double knockdown of CYP99A2 and CYP99A3 specifically suppressed the elicitor-inducible production of momilactones, strongly suggesting that CYP99A2, CYP99A3, or both are involved in momilactone biosynthesis. These results provide strong evidence for the presence on chromosome 4 of a gene cluster involved in momilactone biosynthesis.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Plants that are attacked by pathogenic microorganisms respond with a variety of defense reactions. One such reaction is the production of secondary metabolites that serve as plant antibiotics, known as phytoalexins, which are generated through the perception of signal molecules called elicitors, which are mostly derived from pathogens. Fifteen phytoalexin compounds have been identified in suspension-cultured rice cells treated with biotic elicitors such as a chitin oligosaccharide or a cerebroside (1, 2) and/or from rice leaves that were either infected with the rice leaf blast pathogen Magnaporthe grisea or exposed to UV irradiation (3–10). With the exception of the flavonoid sakuranetin, all of these rice phytoalexins are diterpenoids. These compounds have been classified into four structurally distinct types of polycyclic diterpenoid phytoalexins based on the structures of their diterpene hydrocarbon precursors: phytocassanes A to E, oryzalexins A to F, momilactones A and B, and oryzalexin S. The common precursor geranylgeranyl diphosphate is cyclized to ent-copalyl diphosphate (ent-CDP) and then to ent-cassa-12,15-diene and ent-sandaracopimaradiene, leading to phytocassanes A to E and oryzalexins A to F, respectively. Geranylgeranyl diphosphate is also cyclized to syn-CDP and then to 9betaH-pimara-7,15-diene and stemar-13-ene, leading to momilactones A and B and oryzalexin S, respectively. The hypothetical biosynthetic pathways of the diterpenoid phytoalexins in rice are illustrated in Fig. 1.

We have reported that two type-B diterpene cyclases, ent-CDP synthase (OsCyc2) and syn-CDP synthase (OsCyc1), catalyze the conversion of geranylgeranyl diphosphate to ent-CDP and syn-CDP, respectively (11), and that four type-A diterpene cyclases, ent-cassa-12,15-diene synthase (OsDTC1), ent-sandaracopimaradiene synthase (OsKS10), 9betaH-pimara-7,15-diene synthase (OsKS4), and stemar-13-ene synthase (OsDTC2), catalyze the conversion of ent-CDP or syn-CDP to the four diterpene hydrocarbons ent-cassa-12,15-diene, ent-sandaracopimaradiene, 9betaH-pimara-7,15-diene, and stemar-13-ene, respectively (12–14). The functional identification of OsCyc1 (OsCPSsyn), OsCyc2 (OsCPS2ent), and OsKS4 (OsDTS2) was independently reported by a group at Iowa State University (15–17). OsCyc1, OsCyc2, OsDTC1, and OsDTC2 correspond to OsCPS4, OsCPS2, OsKS7, and OsKS8, respectively, that had been identified as putative rice diterpene cyclase genes by Sakamoto et al. (18).

It has been suggested that microsomal P-450s are involved in the downstream oxidation of the diterpene hydrocarbons, leading to the bioactive phytoalexins by analogy to known biosynthetic pathways for polycyclic diterpenes such as gibberellins (19). In fact, in the biosynthesis of the rice phytoalexins oryzalexins D and E, the conversion of ent-sandaracopimaradien-3beta-ol into oryzalexins D and E was found to be possibly catalyzed by inducible P-450 enzymes, because these enzyme reactions were inhibited by P-450 inhibitors (20). It was also reported that a dehydrogenase is involved in momilactone biosynthesis; a soluble protein fraction from UV-irradiated rice leaves showed dehydrogenase activity, converting 3beta-hydroxy-9betaH-pimara-7,15-dien-19,6beta-olide into momilactone A (21). However, none of the enzymes involved in the downstream oxidation of the diterpene hydrocarbons have been identified.


Figure 1
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  		FIGURE 1.
Proposed pathways for the biosynthesis of diterpenoid phytoalexins in rice.

 
We previously reported that OsDTC1 is located near OsCyc2 on chromosome 2 of the rice genome and that OsKS4 is located near OsCyc1 on chromosome 4 (13). In addition, some P-450 genes have been found near the diterpene cyclase genes on chromosomes 2 and 4, suggesting that the phytocassane and momilactone biosynthesis genes are clustered on chromosomes 2 and 4, respectively (13).

To investigate this hypothesis, in this study, we first focused on the gene cluster on chromosome 4. As described, a dehydrogenase has been suggested to be involved in momilactone biosynthesis. We found that a putative dehydrogenase gene (AK103462) is located near OsKS4, OsCyc1, and two functionally unknown P-450 genes (CYP99A2 and CYP99A3). This information supports the presence of a momilactone biosynthetic gene cluster on chromosome 4. We therefore investigated whether AK103462 and the two P-450 genes (CYP99A2 and CYP99A3) are involved in momilactone biosynthesis.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Chemicals and cDNA Clones—Purified chitooctaose supplied by Yaizu Suisankagaku Industry Co., Ltd. (Tokyo, Japan) was re-N-acetylated to give N-acetylchitooctaose, as described (22), and used as a chitin oligosaccharide elicitor throughout this study. Phytocassanes were isolated from rice leaves infected with M. grisea (8, 9). Momilactones were isolated from rice husks (23), and 3beta-hydroxy-9betaH-pimara-7,15-dien-19,6beta-olide was prepared as described (24). The general P-450 inhibitor uniconazole-P was supplied by Sumitomo Chemical Co. (Takarazuka, Japan).

cDNA clones for AK103462, AK071546 (CYP99A2), and AK071864 (CYP99A3) were provided by the Rice Genome Project of the National Institute of Agrobiological Sciences and the Rice Genome Resource Center in Japan.

Quantification of Diterpenoid Phytoalexins by High Performance Liquid Chromatography-Tandem Mass Spectrometry—An Agilent 1100 separation module (Agilent Technologies, Palo Alto, CA) equipped with a Pegasil C18 column (150 x 2.1 mm in diameter; Senshu Scientific, Tokyo, Japan) was used for HPLC2 analysis. Diterpenoid phytoalexins were eluted in 70% aqueous acetonitrile (MeCN) containing 0.1% acetic acid at a flow rate of 0.2 ml min–1. The respective authentic samples of diterpenoid phytoalexins were dissolved in 79% aqueous ethanol containing 7% MeCN and 0.01% acetic acid for use as standard solutions. For the selection of diagnostic precursor-to-product ion transitions, the standard solutions were directly infused at a flow rate of 5 µl min–1 into a quadrupole tandem mass spectrometer (API 3000; Applied Biosystems Instruments, Foster City, CA) outfitted with an electrospray ion source. All of the diterpenoid phytoalexins were analyzed in positive ion mode. Nitrogen was used as the collision gas. The electrospray capillary was set at 3.0 kV, and the source temperature was 400 °C. Other parameters were optimized using the spectrometer software (Applied Biosystems Instruments). The diterpenoid phytoalexin levels were determined with the m/z combinations (precursor/product ions) of 315/271 for momilactone A, 331/269 for momilactone B, 317/299 for phytocassanes A, D, and E, and 319/301 for phytocassane C in multiple reaction monitoring mode.

Cell Culture and Treatment with Chitin Oligosaccharide Elicitor—Calli of Oryza sativa L. cv. Nipponbare were maintained as described previously (12). Samples of ~1 ml of rice cells were transferred into 100-ml Erlenmeyer flasks containing 30 ml of fresh culture medium (N6 medium with 1 mg l–1 2,4-dichlorophenoxyacetic acid) and cultured on a rotary shaker at 120 rpm and 25 °C, in darkness. After 6 days, the chitin oligosaccharide elicitor was added to the medium to a final concentration of 1 mg l–1. For pretreatment with the P-450 inhibitor uniconazole-P, the compound was added to a final concentration of 30 mg l–1 at 2 h before the addition of the chitin oligosaccharide elicitor.

Quantification of Diterpenoid Phytoalexins in the Rice Cell Culture Medium—To analyze the diterpenoid phytoalexins, 1-ml samples of rice cell culture medium were extracted three times with ethyl acetate (0.5 ml) at approximately pH 5.8. The combined ethyl acetate extracts were concentrated in vacuo. The residues were dissolved in 0.5 ml of 79% aqueous ethanol containing 7% MeCN and 0.01% acetic acid, and 5-µl samples of the resulting solutions were subjected to HPLC-MS/MS.


Figure 2
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  		FIGURE 2.
Scheme of the momilactone biosynthesis gene cluster on rice chromosome 4. The black boxes represent chitin oligosaccharide elicitor-inducible genes that are involved in the biosynthesis of momilactones. The white boxes represent genes that were suggested to be nonresponsive to the chitin oligosaccharide elicitor by microarray analysis (27). No other genes or miRNAs were encoded in the 168-kb region harboring the five elicitor-inducible genes.

 
Expression Analysis of Clustered Genes on Chromosome 4 in Elicited Suspension-cultured Rice Cells and UV-irradiated Rice Leaves—Reverse transcription was performed with SuperScript III RT (Invitrogen) according to the manufacturer's instructions using total RNA (1 µg) that had been prepared from suspension-cultured rice cells (cv. Nipponbare) harvested at 0, 1, 2, 4, 6, 8, 12, or 24 h after the addition of the chitin oligosaccharide elicitor (1 mg l–1). Rice plants (cv. Nipponbare) were cultured in a greenhouse. At the six-leaf stage, the fourth and fifth leaves were detached and UV-irradiated as described previously (12). Control rice leaves (UV–) were handled similarly, except that they were not exposed to UV irradiation. PCR was carried out using KOD-Plus-DNA polymerase (Toyobo) with the following program: 2 min at 94 °C followed by 35 cycles of 15 s at 94 °C, 30 s at 55 °C, and 1 min 50 s at 68 °C, followed by cooling to 4 °C. The pairs of gene-specific primers that were used are as follows: for CYP99A2, 5'-CTACTCACCAATAATCCATCCTCG-3' (forward) and 5'-CATTACGTGCACCCTTACAAGG-3' (reverse); for CYP99A3, 5'-GCTAAACTGCCAAGTAATTAAAC-3' (forward) and 5'-GAGGTATGCAAGCACGTAAG-3' (reverse); for AK103462, 5'-CCATGTTTCTGCTGATGCAA-3' (forward) and 5'-TCCCTGAAGAAACCAAAGCTC-3' (reverse); and for the rice actin gene ACT1 as an internal standard, 5'-CATGCTATCCCTCGTCTCGACCT-3' (forward) and 5'-CGCACTTCATGATGGAGTTGTAT-3' (reverse).

Expression of the AK103462 cDNA in Escherichia coli—The AK103462 ORF was subcloned into pENTR/D-TOPO vector using the Gateway pENTR/D-TOPO cloning kit (Invitrogen). The resulting entry clone was used for recombination of the Gateway expression vector pDEST17 (Invitrogen) to add an N-terminal His6 tag. The resulting plasmid (pDEST17-AK103462) was transformed into the E. coli strain Rosetta (DE3) (Invitrogen). The strain was grown at 37 °C for 10 h in LB medium (10 g l–1 tryptone, 5 g l–1 yeast extract, 5 g l–1 NaCl, pH 7.0) containing 50 µgml–1 ampicillin. The collected cells were resuspended in 20 mM Tris-HCl and disrupted by mild sonication on ice. After centrifugation at 3500 x g for 15 min, the supernatant was affinity-purified using the QuickPickTM IMAC Plus kit (Nacalai Tesque, Kyoto, Japan) to yield His6-tagged OsMAS. As a control, E. coli strain Rosetta (DE3) was similarly cultured, and the bacterial lysate was subjected to affinity column chromatography. The affinity-purified His6-tagged AK103462 protein was analyzed by SDS-PAGE in a 10% acrylamide gel followed by staining with Coomassie Brilliant Blue.

Enzyme Assay of His6-tagged AK103462 Protein—A reaction mixture was prepared containing 10 µg of His6-tagged AK103462 protein and 500 µl of assay buffer consisting of 0.2 M Tris-HCl, pH 8.0, 10 mM 2-mercaptoethanol, 1 mM NAD+, and 10 µg 3beta-hydroxy-9betaH-pimara-7,15-dien-19,6beta-olide. After incubation at 30 °C for 40 min, the reaction was stopped by the addition of 0.5 ml of methanol. The mixture was then extracted with ethyl acetate, and the extract was evaporated to dryness in vacuo. The residue was dissolved in 500 µl of 79% aqueous ethanol containing 7% MeCN and 0.01% acetic acid, and a 5-µl sample of the solution was subjected to HPLC-MS/MS. The ethyl acetate extract from the reaction mixture was also subjected to gas chromatography mass spectrometry, which was conducted using an Agilent 6890N JEOL K9 mass selective detector system (ionization voltage 70 eV) fitted with a fused silica chemically bonded capillary column (DB-5; 0.25 mm in diameter, 15 m long, 0.25-µm film thickness; J&W Scientific Inc., Folsom, CA). Each sample was injected onto the column at 120 °C in the splitless mode. After a 2-min isothermal hold at 120 °C, the column temperature was increased by 16 °C min–1 to 280 °C with a 3-min isothermal hold at 280 °C. The flow rate of the helium carrier gas was 1 ml min–1.

Double Knockdown of CYP99A2 and CYP99A3 by RNA Interference (RNAi)—A CYP99A2-specific RNAi plasmid was constructed using a Gateway destination vector, pANDA (25), which was kindly supplied by Drs. D. Miki and K. Shimamoto (Nara Institute of Science and Technology, Ikoma, Japan). A CYP99A2 cDNA fragment (325 bp) consisting of a 108-bp 3'-UTR and a 217-bp 3'-terminal region of the ORF was synthesized as an RNAi trigger region by PCR. The pair of gene-specific primers are as follows: 5'-CACCGTTTGGAACCGGGAGGAGG-3' (forward) and 5'-ACCCTTACAAGGTACACAAACACGC-3' (reverse). Subcloning of the PCR product with the pENTR/DTOPO vector was carried out according to the manufacturer's instructions. The plasmid pANDA-CYP99A2-RNAi was constructed by recombination reaction of the entry clone (pENTR-CYP99A2) and the pANDA vector with the Gateway LR Clonase enzyme mix (Invitrogen). The pANDA-CYP99A2-RNAi vector was used to generate transgenic rice cells (var. Nipponbare) by Agrobacterium-mediated transformation (26). Double knockdown of CYP99A2 and CYP99A3 expression was confirmed by RT-PCR as described above.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Characterization of a 168-kb Gene Cluster on Chromosome 4—Utilizing the Rice Genome Automated Annotation System (RiceGAAS; ricegaas.dna.affrc.go.jp/), we found that the genes OsCyc1, CYP99A3, the putative dehydrogenase gene AK103462, OsKS4, and CYP99A2 are linearly arranged within a 168-kb region on chromosome 4 (Fig. 2). No other genes or miRNAs were encoded in this 168-kb region. The transcription of OsCyc1 and OsKS4 is up-regulated in suspension-cultured rice cells treated with a chitin oligosaccharide elicitor (18, 27) and in rice leaves irradiated with UV light (11, 13, 18). We therefore performed semiquantitative RT-PCR analysis on AK103462, CYP99A2, and CYP99A3 in rice cells treated with the chitin oligosaccharide elicitor N-acetylchitooctaose and in UV-irradiated rice leaves. As shown in Fig. 3, the expression of all three genes was up-regulated in both the elicited rice cells and the UV-irradiated leaves. The AK103462 cDNA is 1265 bp in length, with an ORF encoding 274 amino acid residues. The ORF shows 55.0% identity to secoisolariciresinol dehydrogenase of Forsythia intermedia (28). The CYP99A2 and CYP99A3 cDNAs are 1780 and 1750 bp in length with ORFs encoding 502 and 507 amino acid residues, respectively. The amino acid sequences encoded by CYP99A2 and CYP99A3 share 55.8 and 55.0% identities with that encoded by CYP99A1, respectively, which is a functionally unknown P-450 identified in Sorghum bicolor (29). The CYP99A2 and CYP99A3 amino acid sequences are 83.9% identical, and no other P-450 ORFs with greater than 40% identity to these ORFs are present in the rice genome.


Figure 3
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  		FIGURE 3.
Expression analysis of AK103462, CYP99A2, and CYP99A3 in rice cells treated with the chitin oligosaccharide elicitor (a) and in UV-irradiated rice leaves (b). RT-PCR was performed using gene-specific primers and total RNA (1µg) prepared from rice cells treated with the elicitor (1 mg l–1) for the indicated times and UV-irradiated (+) and control (–) rice leaves. As an internal standard, the rice actin gene ACT1 was amplified by RT-PCR using gene-specific primers.

 
Functional Analysis of the AK103462 Protein—A soluble fraction from UV-irradiated rice leaves has shown dehydrogenase activity that catalyzes the conversion of 3beta-hydroxy-9betaH-pimara-7,15-dien-19,6beta-olide into momilactone A (21). Considering the possibility that the putative dehydrogenase gene AK103462, together with OsCyc1 and OsKS4, is involved in momilactone biosynthesis, we investigated whether the AK103462 gene encodes momilactone A synthase.

The AK103462 protein was overexpressed in E. coli as a His6-tagged fusion protein. The E. coli strain Rosetta (DE3), which harbors a plasmid that directs the expression of the His6-tagged fusion protein, was used, and the bacterial lysate was subjected to affinity column chromatography and then to SDS-PAGE to confirm production of the fusion protein.

Enzyme assays of the affinity-purified His6-tagged fusion protein for momilactone A synthase activity were performed using 3beta-hydroxy-9betaH-pimara-7,15-dien-19,6beta-olide as a substrate, and the reaction product was analyzed by HPLC-MS/MS. As shown in Fig. 4a, it was indicated that the substrate was efficiently converted into momilactone A by the recombinant protein. The identification of momilactone A as a reaction product was further confirmed by gas chromatography MS analysis as follows: the reaction product (retention time (Rt)11 min 0 s), m/z (% base peak) 314 (M+, 24%), 299 (9), 286 (2), 271 (24), 255 (31), 241 (9), 227 (17), 213 (33), 199 (100), 187 (30), 173 (26), 157 (31), 145 (39), 133 (45), 119 (33), 105 (45), 91 (77), 81 (91), 67 (41), and 55 (48); authentic momilactone A (Rt 11 min 1 s), m/z (% base peak) 314 (M+, 23%), 299 (8), 286 (2), 271 (23), 255 (32), 241 (9), 227 (18), 213 (32), 199 (100), 187 (30), 173 (27), 157 (31), 145 (40), 133 (44), 119 (32), 105 (43), 91 (76), 81 (85), 67 (40), and 55 (47). In contrast, in the control experiment, only the substrate was recovered (Fig. 4b). These results demonstrate that the AK103462 gene encodes momilactone A synthase. Therefore, this gene has been named the O. sativa L. momilactone A synthase gene (OsMAS).


Figure 4
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  		FIGURE 4.
HPLC-MS/MS analysis of the product generated by the incubation of the substrate 3beta-hydroxy-9betaH-pimara-7,15-dien-19,6beta-olide with the recombinant AK103462 protein. a, AK103462 protein with substrate; b, control preparation with substrate. Using HPLC-MS/MS, the contents of momilactone A and 3beta-hydroxy-9betaH-pimara-7,15-dien-19,6beta-olide were determined using the m/z combinations 315/271 and 317/299, respectively, in multiple reaction monitoring mode.

 
Involvement of CYP99A2 and CYP99A3 in Momilactone Biosynthesis—We next investigated whether the P-450 genes CYP99A2 and CYP99A3 in the 168-kb gene cluster on chromosome 4 are involved in momilactone biosynthesis.

We treated suspension-cultured rice cells with uniconazole-P, a P-450 inhibitor, which was followed 2 h later with treatment with the chitin oligosaccharide elicitor. At 48 h after the elicitor treatment, we determined the levels of the major diterpenoid phytoalexins momilactones and phytocassanes in the culture medium using HPLC-MS/MS. As shown in Fig. 5, the chitin oligosaccharide elicitor-inducible production of momilactones A and B and phytocassanes A to E was much lower than in rice cells that had not been treated with uniconazole-P. These results indicate that P-450s might be involved in the biosynthesis of momilactones and phytocassanes.


Figure 5
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  		FIGURE 5.
Effects of the P-450 inhibitor uniconazole-P on the production of phytoalexins. The amounts of momilactones A and B and phytocassanes A to E that accumulated in rice cell culture medium at 48 h after treatment with 1 mg l–1 N-acetylchitooctaose were determined by HPLC-MS/MS. Pretreatment with (+) or without (–) the P-450 inhibitor uniconazole-P at a final concentration of 30 mg l–1 was carried out 2 h before the addition of the N-acetylchitooctaose elicitor. The quantification was carried out two times with reproducible results. One series of data is shown. MA, MB, PA, PB, PC, PD, and PE indicate momilactones A and B and phytocassanes A, B, C, D, and E, respectively.

 


Figure 6
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  		FIGURE 6.
Double knockdown of CYP99A2 and CYP99A3. The expression of CYP99A2 and CYP99A3 was examined by RT-PCR in double-knockdown cell lines at 12 h after treatment with 1 mg l–1 N-acetylchitooctaose (a). As an internalstandard, ACT1was amplified by RT-PCR using gene-specific primers. The amounts of momilactone A (b), momilactone B (c), or phytocassanes A, B, C, D, and E(d) that accumulated in culture medium of the double-knockdown celllines at 48 h after treatment with 1 mg l–1 N-acetylchitooctaose were determined by HPLC-MS/MS. The results are expressed as the means±S.E. of three experiments.

 
To examine the involvement of CYP99A2 and CYP99A3 in the chitin oligosaccharide elicitor-inducible production of diterpenoid phytoalexins in rice cells, RNAi-mediated knockdown of CYP99A2 and CYP99A3 was attempted. The 3'-UTRs of CYP99A2 and CYP99A3 are 162 and 202 bp long, and they show 33.9% identity to each other, whereas the ORFs of the two genes share 87% identity at the nucleotide sequence level (83.9% identity at the amino acid sequence level). To generate RNAi constructs for gene suppression, it is preferable to generate 300–500-bp fragments of gene sequences by PCR from genes of interest (25). We therefore used a 325-bp region as an input trigger for RNAi knockdown of CYP99A2. Thus, the input trigger consisted of a 108-bp 3'-UTR and a 217-bp 3'-terminal region of the ORF, which is highly homologous to that of CYP99A3. RNAi-mediated knockdown of CYP99A2 using this input trigger resulted in production of the double knockdown of the two genes (Fig. 6a). We also attempted RNAi-mediated knockdown of CYP99A3 using the plasmid harboring the trigger region consisting of an 81-bp 3'-UTR and a 207-bp 3'-terminal region of the CYP99A3 ORF, resulting in production of the double knockdown lines (data not shown).

The accumulation of momilactones and phytocassanes in rice cell culture medium was determined using HPLC-MS/MS. As shown in Fig. 6 (b–d), in culture medium of the double-knockdown cell lines 1 and 2 at 48 h after chitin oligosaccharide elicitor treatment, the accumulation of momilactones A and B was much lower than in control medium, but total amounts of phytocassanes that accumulated in the knockdown lines and control medium were similar.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
In this study, we investigated the function of a 168-kb gene cluster on rice chromosome 4 that consists of a possible dehydrogenase gene (AK103462), two functionally unknown P-450 genes (CYP99A2 and CYP99A3), and two diterpene cyclase genes (OsCyc1 and OsKS4) involved in momilactone biosynthesis (23) (Fig. 2). Semiquantitative RT-PCR revealed that the AK103462 gene, CYP99A2, and CYP99A3 are inducible by chitin oligosaccharide elicitor treatment in suspension-cultured rice cells and by UV irradiation in rice leaves (Fig. 3). Because the diterpene cyclase genes OsCyc1 and OsKS4 are also known to be chitin oligosaccharide elicitor- and UV-inducible (11, 13, 23), these results suggest that AK103462, CYP99A2, and CYP99A3, together with OsCyc1 and OsKS4, form a chitin oligosaccharide elicitor- and UV irradiation-inducible gene cluster on chromosome 4 and that they are involved in phytoalexin biosynthesis.

We next carried out functional analysis of the AK103462 protein by heterologous expression in E. coli followed by enzyme assays and demonstrated that the AK103462 protein (OsMAS) functions as momilactone A synthase, catalyzing the conversion of 3beta-hydroxy-9betaH-pimara-7,15-dien-19,6beta-olide into momilactone A (Fig. 4).

We further attempted to perform knockdown experiments using plasmids for the gene-specific knockdowns of CYP99A2 and CYP99A3. However, the knockdown of either gene resulted in the production of double-knockdown lines of both. As already described, this is because the input trigger consisted of not only a gene-specific region from the 3'-UTR but a 3'-terminal region of the ORF. Because the nucleotide sequences of CYP99A2 and CYP99A3 are highly similar to each other, siRNAs directed against either gene could target both the CYP99A2 and CYP99A3 ORFs. In the double-knockdown lines, momilactone biosynthesis was specifically suppressed (Fig. 6, a–c), strongly suggesting that CYP99A2, CYP99A3, or both are involved in biosynthetic steps between 9betaH-pimara-7,15-diene and 3beta-hydroxy-9betaH-pimara-7,15-dien-19,6beta-olide in the momilactone biosynthetic pathway (Fig. 7). These results provide strong evidence for the presence of a gene cluster consisting of four or five genes that are involved in the biosynthesis of momilactones on chromosome 4. In eukaryotes, gene clusters for the synthesis of secondary metabolites, such as gibberellins, aphidicolin, and abscisic acid, have been reported in fungi (30–32). In higher plants, it was reported that five genes involved in biosynthesis of the cyclic hydroxamic acid 2,4-dihydroxy-7-methoxy-1,4-benzoxazin-3-one are localized to a 6-cM region on the short arm of chromosome 4 in maize. 2,4-Dihydroxy-7-methoxy-1,4-benzoxazin-3-one is a secondary metabolite that forms part of the defense against insects and microbial pathogens (33).


Figure 7
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  		FIGURE 7.
Involvement of CYP99A2, CYP99A3, and OsMAS in the downstream oxidation of 9betaH-pimara-7,15-diene, leading to the momilactones A and B.

 
It is also noteworthy that all of the momilactone biosynthetic genes clustered on chromosome 4 are coordinately expressed after treatment with a chitin oligosaccharide elicitor. Coordinate elicitor-inducible expression of this cluster of genes might suggest the presence of common cis-elements in the promoter regions of the respective genes. Computational analysis of the promoter regions indicates that the respective genes contain recognition sites of transcriptional factors such as WRKY and basic leucine zipper (bZIP) proteins that have been indicated to be involved defense responses (34, 35). To elucidate regulatory mechanisms for the coordinate expression of this gene cluster of genes, promoter analysis followed by identification of transcription factors involved in expression of the respective genes will be required. We have previously suggested that a phytocassane biosynthetic gene cluster is present on chromosome 2 (13). Momilactones and phytocassanes are major representatives of four distinct types of diterpenoid phytoalexins in rice. Although the biological significance of gene clusters for the synthesis of secondary metabolites is not clear, such gene clusters might contribute to the efficient coordinated expression of the genes by elicitation, followed by the production of high levels of diterpenoid phytoalexins.


   FOOTNOTES
 
* This work was supported in part by Grants-in-Aid for Scientific Research 16208012 and 18580102 from the Japanese Society for the Promotion of Science and by the Program for Promotion of Basic Research Activities for Innovative Biosciences. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

1 To whom correspondence should be addressed. Fax: 81-3-5841-8030; E-mail: ayamane{at}mail.ecc.u-tokyo.ac.jp.

2 The abbreviations used are: HPLC, high performance liquid chromatography; MS/MS, tandem mass spectrometry; ORF, open reading frame; RNAi, RNA interference; UTR, untranslated region; RT, reverse transcription. Back



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 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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