Identification of a Biosynthetic Gene Cluster in Rice for Momilactones
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 3β-hydroxy-9βH-pimara-7,15-dien-19,6β-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. 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 3β-hydroxy-9βH-pimara-7,15-dien-19,6β-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. 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 (1Yamada A. Shibuya N. Komada O. Akatsuka T. Biosci. Biotechnol. Biochem. 1993; 57: 405-409Crossref Scopus (250) Google Scholar, 2Umemura K. Ogawa N. Koga J. Iwata M. Usami H. Plant Cell Physiol. 2002; 43: 778-784Crossref PubMed Scopus (65) Google Scholar) and/or from rice leaves that were either infected with the rice leaf blast pathogen Magnaporthe grisea or exposed to UV irradiation (3Cartwright D.W. Langcake P.W. Pryce R.J. Leworthy D.P. Ride J.P. Phytochemistry. 1981; 20: 535-537Crossref Scopus (180) Google Scholar, 4Akatsuka T. Takahashi N. Kodama O. Sekido H. Kono Y. Takeuchi S. Agric. Biol. Chem. 1985; 49: 1689-1694Crossref Scopus (67) Google Scholar, 5Kato H. Kodama O. Akatsuka T. Phytochemistry. 1993; 33: 79-81Crossref Scopus (90) Google Scholar, 6Kato H. Kodama O. Akatsuka T. Phytochemistry. 1994; 36: 299-301Crossref Scopus (86) Google Scholar, 7Tamogami S. Mitani M. Kodama O. Akatsuka T. Tetrahedron. 1993; 49: 2025-2032Crossref Scopus (62) Google Scholar, 8Koga J. Shimura M. Oshima K. Ogawa N. Yamauchi T. Ogasawara N. Tetrahedron. 1995; 51: 7907-7918Crossref Scopus (102) Google Scholar, 9Koga J. Ogawa N. Yamauchi T. Kikuchi M. Ogasawara N. Shimura M. Phytochemistry. 1997; 44: 249-253Crossref Scopus (92) Google Scholar, 10Kodama O. Miyakawa J. Akatsuka T. Kiyosawa S. Phytochemistry. 1992; 31: 3807-3809Crossref Scopus (226) Google Scholar). 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 9βH-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 (11Otomo K. Kenmoku H. Oikawa H. Konig W.A. Toshima H. Mitsuhashi W. Yamane H. Sassa T. Toyomasu T. Plant J. 2004; 39: 886-893Crossref PubMed Scopus (140) Google Scholar), and that four type-A diterpene cyclases, ent-cassa-12,15-diene synthase (OsDTC1), ent-sandaracopimaradiene synthase (OsKS10), 9βH-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, 9βH-pimara-7,15-diene, and stemar-13-ene, respectively (12Cho E.-M. Okada A. Kenmoku H. Otomo K. Toyomasu T. Mitsuhashi W. Sassa T. Yajima A. Yabuta G. Mori K. Oikawa H. Toshima H. Shibuya N. Nojiri H. Omori T. Nishiyama M. Yamane H. Plant J. 2004; 37: 1-8Crossref PubMed Scopus (89) Google Scholar, 13Otomo K. Kanno Y. Motegi A. Kenmoku H. Yamane H. Mitsuhashi W. Oikawa H. Toshima H. Itoh H. Matsuoka M. Sassa T. Toyomasu T. Biosci. Biotechnol. Biochem. 2004; 68: 2001-2006Crossref PubMed Scopus (82) Google Scholar, 14Nemoto T. Cho E.-M. Okada A. Okada K. Otomo K. Kanno Y. Toyomasu T. Mitsuhashi W. Sassa T. Minami E. Shibuya N. Nishiyama M. Nojiri H. Yamane H. FEBS Lett. 2004; 571: 182-186Crossref PubMed Scopus (58) Google Scholar). The functional identification of OsCyc1 (OsCPSsyn), OsCyc2 (OsCPS2ent), and OsKS4 (OsDTS2) was independently reported by a group at Iowa State University (15Xu M. Hillwig M.L. Prisic S. Coates R.M. Peters R.J. Plant J. 2004; 39: 309-318Crossref PubMed Scopus (126) Google Scholar, 16Prisic S. Xu M. Wilderman P.R. Peters R.J. Plant Physiol. 2004; 136: 4228-4236Crossref PubMed Scopus (136) Google Scholar, 17Wilderman P.R. Xu M. Jin Y. Coates R.M. Peters R.J. Plant Physiol. 2004; 135: 2098-2105Crossref PubMed Scopus (163) Google Scholar). 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. (18Sakamoto T. Miura K. Itoh H. Tatsumi T. Ueguchi-Tanaka M. Ishiyama K. Kobayashi M. Agrawal G.K. Takeda S. Abe K. Miyao A. Hirochika H. Kitano H. Ashikari M. Matsuoka M. Plant Physiol. 2004; 134: 1642-1653Crossref PubMed Scopus (525) Google Scholar). 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 (19Peters R.J. Phytochemistry. 2006; 67: 2307-2317Crossref PubMed Scopus (150) Google Scholar). In fact, in the biosynthesis of the rice phytoalexins oryzalexins D and E, the conversion of ent-sandaracopimaradien-3β-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 (20Kato H. Kodama O. Akatsuka T. Arch. Biochem. Biophys. 1995; 316: 707-712Crossref PubMed Scopus (25) Google Scholar). 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 3β-hydroxy-9βH-pimara-7,15-dien-19,6β-olide into momilactone A (21Atawong A. Hasegawa M. Kodama O. Biosci. Biotechnol. Biochem. 2002; 66: 566-570Crossref PubMed Scopus (34) Google Scholar). However, none of the enzymes involved in the downstream oxidation of the diterpene hydrocarbons have been identified. 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 (13Otomo K. Kanno Y. Motegi A. Kenmoku H. Yamane H. Mitsuhashi W. Oikawa H. Toshima H. Itoh H. Matsuoka M. Sassa T. Toyomasu T. Biosci. Biotechnol. Biochem. 2004; 68: 2001-2006Crossref PubMed Scopus (82) Google Scholar). 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 (13Otomo K. Kanno Y. Motegi A. Kenmoku H. Yamane H. Mitsuhashi W. Oikawa H. Toshima H. Itoh H. Matsuoka M. Sassa T. Toyomasu T. Biosci. Biotechnol. Biochem. 2004; 68: 2001-2006Crossref PubMed Scopus (82) Google Scholar). 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. 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 (22Ito Y. Kaku H. Shibuya N. Plant J. 1997; 12: 347-356Crossref PubMed Scopus (131) Google Scholar), and used as a chitin oligosaccharide elicitor throughout this study. Phytocassanes were isolated from rice leaves infected with M. grisea (8Koga J. Shimura M. Oshima K. Ogawa N. Yamauchi T. Ogasawara N. Tetrahedron. 1995; 51: 7907-7918Crossref Scopus (102) Google Scholar, 9Koga J. Ogawa N. Yamauchi T. Kikuchi M. Ogasawara N. Shimura M. Phytochemistry. 1997; 44: 249-253Crossref Scopus (92) Google Scholar). Momilactones were isolated from rice husks (23Kato T. Tsunakawa M. Sasaki N. Aizawa H. Fujita K. Kitahara Y. Takahashi N. Phytochemistry. 1977; 16: 45-48Crossref Scopus (145) Google Scholar), and 3β-hydroxy-9βH-pimara-7,15-dien-19,6β-olide was prepared as described (24Kato T. Aizawa H. Tsunakawa M. Sasaki N. Kitahara Y. Takahashi N. J. Chem. Soc. Perkin I. 1977; : 250-254Crossref Scopus (22) Google Scholar). 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 × 2.1 mm in diameter; Senshu Scientific, Tokyo, Japan) was used for HPLC 2The 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. 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 of were as described previously (12Cho E.-M. Okada A. Kenmoku H. Otomo K. Toyomasu T. Mitsuhashi W. Sassa T. Yajima A. Yabuta G. Mori K. Oikawa H. Toshima H. Shibuya N. Nojiri H. Omori T. Nishiyama M. Yamane H. Plant J. 2004; 37: 1-8Crossref PubMed Scopus (89) Google Scholar). of ml of rice cells were into containing ml of with and on a at and in the chitin oligosaccharide elicitor was to the to a of For with the P-450 inhibitor the was to a of at 2 the of the chitin oligosaccharide Quantification of Diterpenoid Phytoalexins in the Rice Cell Culture the diterpenoid phytoalexins, samples of rice were with at The were in The were dissolved in ml of 79% aqueous ethanol containing 7% MeCN and 0.01% acetic and samples of the solutions were to of on 4 in Rice and UV-irradiated Rice was with to the using RNA that had been prepared from suspension-cultured rice cells at 4, or the of the chitin oligosaccharide elicitor Rice were in a the the and leaves were and UV-irradiated as described previously (12Cho E.-M. Okada A. Kenmoku H. Otomo K. Toyomasu T. Mitsuhashi W. Sassa T. Yajima A. Yabuta G. Mori K. Oikawa H. Toshima H. Shibuya N. Nojiri H. Omori T. Nishiyama M. Yamane H. Plant J. 2004; 37: 1-8Crossref PubMed Scopus (89) Google Scholar). rice leaves were that were exposed to UV was using with the 2 at followed by of at at and at followed by to 4 °C. The of that were used are as for CYP99A2, and for CYP99A3, and for AK103462, and and for the rice gene as an and of the AK103462 cDNA in Escherichia AK103462 was into using the The was used for of the expression to an The was into the E. coli The was at for in 5 5 containing The cells were in mm and by on at × for the was using the Japan) to As a E. coli was and the was to column The AK103462 protein was analyzed by in a followed by with of AK103462 reaction was prepared containing of AK103462 protein and μl of of 0.2 mm mm and at for the reaction was by the of ml of The was then with and the was to in The was dissolved in μl of 79% aqueous ethanol containing 7% MeCN and 0.01% acetic and a of the was to The from the reaction was also to mass which was using an Agilent mass with a capillary column mm in was the column at in the mode. a at the column temperature was by min–1 to with a at °C. The flow rate of the was ml min–1. of CYP99A2 and CYP99A3 by RNA was using a K. Plant Cell Physiol. 2004; PubMed Scopus Google Scholar), which was supplied by and K. Institute of and Japan). A CYP99A2 cDNA of a and a of the was as an by The of are as and of the with the was to the The was by reaction of the and the with the enzyme The was used to rice cells by H. Y. N. N. K. Minami E. Shibuya N. S. A. 2006; PubMed Scopus Google Scholar). knockdown of CYP99A2 and CYP99A3 expression was by as described of a on the Rice Genome we found that the genes OsCyc1, CYP99A3, the putative dehydrogenase gene AK103462, OsKS4, and CYP99A2 are a on chromosome 4 genes or were in this The of OsCyc1 and OsKS4 is in suspension-cultured rice cells treated with a chitin oligosaccharide elicitor (18Sakamoto T. Miura K. Itoh H. Tatsumi T. Ueguchi-Tanaka M. Ishiyama K. Kobayashi M. Agrawal G.K. Takeda S. Abe K. Miyao A. Hirochika H. Kitano H. Ashikari M. Matsuoka M. Plant Physiol. 2004; 134: 1642-1653Crossref PubMed Scopus (525) Google Scholar, A. T. Okada K. T. Koga J. Shibuya N. Nojiri H. Yamane H. Plant Biol. PubMed Scopus Google Scholar) and in rice leaves irradiated with UV (11Otomo K. Kenmoku H. Oikawa H. Konig W.A. Toshima H. Mitsuhashi W. Yamane H. Sassa T. Toyomasu T. Plant J. 2004; 39: 886-893Crossref PubMed Scopus (140) Google Scholar, 13Otomo K. Kanno Y. Motegi A. Kenmoku H. Yamane H. Mitsuhashi W. Oikawa H. Toshima H. Itoh H. Matsuoka M. Sassa T. Toyomasu T. Biosci. Biotechnol. Biochem. 2004; 68: 2001-2006Crossref PubMed Scopus (82) Google Scholar, T. Miura K. Itoh H. Tatsumi T. Ueguchi-Tanaka M. Ishiyama K. Kobayashi M. Agrawal G.K. Takeda S. Abe K. Miyao A. Hirochika H. Kitano H. Ashikari M. Matsuoka M. Plant Physiol. 2004; 134: 1642-1653Crossref PubMed Scopus (525) Google Scholar). We therefore analysis on AK103462, CYP99A2, and CYP99A3 in rice cells treated with the chitin oligosaccharide elicitor and in UV-irradiated rice As in Fig. the expression of all genes was in both the rice cells and the UV-irradiated The AK103462 cDNA is in with an acid The to dehydrogenase of J. Biol. Chem. PubMed Scopus Google Scholar). The CYP99A2 and CYP99A3 are and in with and acid respectively. The acid by CYP99A2 and CYP99A3 and with that by respectively, which is a functionally unknown P-450 identified in S. Plant Biol. 36: PubMed Scopus Google Scholar). The CYP99A2 and CYP99A3 acid are and P-450 with to these are in the rice analysis of AK103462, CYP99A2, and CYP99A3 in rice cells treated with the chitin oligosaccharide elicitor and in UV-irradiated rice leaves was using and RNA prepared from rice cells treated with the elicitor for the and UV-irradiated and rice As an the rice gene was by using Functional of the AK103462 soluble fraction from UV-irradiated rice leaves has dehydrogenase that catalyzes the conversion of 3β-hydroxy-9βH-pimara-7,15-dien-19,6β-olide into momilactone A (21Atawong A. Hasegawa M. Kodama O. Biosci. Biotechnol. Biochem. 2002; 66: 566-570Crossref PubMed Scopus (34) Google Scholar). the that the putative dehydrogenase gene AK103462, together with OsCyc1 and OsKS4, is involved in momilactone we investigated whether the AK103462 gene momilactone A The AK103462 protein was in E. coli as a The E. coli which a that the expression of the was and the was to column and then to to production of the assays of the protein for momilactone A synthase were using 3β-hydroxy-9βH-pimara-7,15-dien-19,6β-olide as a and the reaction was analyzed by As in Fig. was that the substrate was converted into momilactone A by the The identification of momilactone A as a reaction was by analysis as the reaction m/z and authentic momilactone A m/z and In in the the substrate was These results that the AK103462 gene momilactone A this gene has been the O. momilactone A synthase gene of CYP99A2 and CYP99A3 in investigated whether the P-450 genes CYP99A2 and CYP99A3 in the gene cluster on chromosome 4 are involved in momilactone biosynthesis. We treated suspension-cultured rice cells with a P-450 which was followed 2 with with the chitin oligosaccharide the elicitor we determined the levels of the diterpenoid phytoalexins momilactones and phytocassanes in the using As in Fig. the chitin oligosaccharide elicitor-inducible production of momilactones A and B and phytocassanes A to E was in rice cells that had been treated with These results that P-450s be involved in the biosynthesis of momilactones and To the of CYP99A2 and CYP99A3 in the chitin oligosaccharide elicitor-inducible production of diterpenoid phytoalexins in rice knockdown of CYP99A2 and CYP99A3 was The of CYP99A2 and CYP99A3 are and and to the of the two genes at the at the acid To for gene is to of gene by from genes of K. Plant Cell Physiol. 2004; PubMed Scopus Google Scholar). We therefore used a as an for knockdown of the of a and a of the ORF, which is to that of knockdown of CYP99A2 using this in production of the double knockdown of the two genes We also knockdown of CYP99A3 using the the of an and a of the CYP99A3 ORF, in production of the double knockdown The of momilactones and phytocassanes in rice was determined using As in Fig. in of the and 2 at chitin oligosaccharide elicitor the of momilactones A and B was in of phytocassanes that in the knockdown and were In this study, we investigated the of a gene cluster on rice chromosome 4 that of a dehydrogenase gene two functionally unknown P-450 genes (CYP99A2 and and two diterpene cyclase genes and involved in momilactone biosynthesis (23Kato T. Tsunakawa M. Sasaki N. Aizawa H. Fujita K. Kitahara Y. Takahashi N. Phytochemistry. 1977; 16: 45-48Crossref Scopus (145) Google Scholar) that the AK103462 CYP99A2, and CYP99A3 are inducible by chitin oligosaccharide elicitor in suspension-cultured rice cells and by UV irradiation in rice leaves the diterpene cyclase genes OsCyc1 and OsKS4 are also known to be chitin oligosaccharide elicitor- and UV-inducible (11Otomo K. Kenmoku H. Oikawa H. Konig W.A. Toshima H. Mitsuhashi W. Yamane H. Sassa T. Toyomasu T. Plant J. 2004; 39: 886-893Crossref PubMed Scopus (140) Google Scholar, 13Otomo K. Kanno Y. Motegi A. Kenmoku H. Yamane H. Mitsuhashi W. Oikawa H. Toshima H. Itoh H. Matsuoka M. Sassa T. Toyomasu T. Biosci. Biotechnol. Biochem. 2004; 68: 2001-2006Crossref PubMed Scopus (82) Google Scholar, T. Tsunakawa M. Sasaki N. Aizawa H. Fujita K. Kitahara Y. Takahashi N. Phytochemistry. 1977; 16: 45-48Crossref Scopus (145) Google Scholar), these results that AK103462, CYP99A2, and CYP99A3, together with OsCyc1 and OsKS4, form a chitin oligosaccharide elicitor- and UV gene cluster on chromosome 4 and that are involved in phytoalexin biosynthesis. We functional analysis of the AK103462 protein by heterologous expression in E. coli followed by enzyme assays and demonstrated that the AK103462 protein as momilactone A the conversion of 3β-hydroxy-9βH-pimara-7,15-dien-19,6β-olide into momilactone A We to knockdown using for the of CYP99A2 and However, the knockdown of either gene in the production of of As described, this is because the of a from the a of the the of CYP99A2 and CYP99A3 are to either gene both the CYP99A2 and CYP99A3 In the momilactone biosynthesis was specifically suppressed strongly suggesting that CYP99A2, CYP99A3, or both are involved in biosynthetic 9βH-pimara-7,15-diene and 3β-hydroxy-9βH-pimara-7,15-dien-19,6β-olide in the momilactone biosynthetic These results provide strong evidence for the presence of a gene cluster of four or genes that are involved in the biosynthesis of momilactones on chromosome 4. In gene for the of secondary such as and have been reported in E. 67: PubMed Scopus (82) Google Scholar, T. K. Toshima H. T. K. H. H. Mitsuhashi W. Sassa T. Oikawa H. Biosci. Biotechnol. Biochem. 2004; 68: PubMed Scopus Google Scholar, J. 2006; PubMed Scopus Google Scholar). In was reported that genes involved in biosynthesis of the acid are to a on the of chromosome 4 in is a secondary that of the defense and M. E. A. A. W. A. K. A. 1997; PubMed Scopus Google Scholar). It is also that all of the momilactone biosynthetic genes clustered on chromosome 4 are with a chitin oligosaccharide elicitor-inducible expression of this cluster of genes the presence of common in the of the respective analysis of the that the respective genes of such as and that have been to be involved defense T. S. Minami E. Y. Kaku H. Shibuya N. M. H. K. Okada A. Okada K. Nojiri H. Yamane H. Biophys. PubMed Scopus Google Scholar, E. Plant Biol. 1997; PubMed Scopus Google Scholar). To for the expression of this gene cluster of analysis followed by identification of involved in expression of the respective genes be We have previously suggested that a phytocassane biosynthetic gene cluster is on chromosome 2 (13Otomo K. Kanno Y. Motegi A. Kenmoku H. Yamane H. Mitsuhashi W. Oikawa H. Toshima H. Itoh H. Matsuoka M. Sassa T. Toyomasu T. Biosci. Biotechnol. Biochem. 2004; 68: 2001-2006Crossref PubMed Scopus (82) Google Scholar). Momilactones and phytocassanes are of four distinct types of diterpenoid phytoalexins in the of gene for the of secondary metabolites is such gene to the expression of the genes by followed by the production of high levels of diterpenoid