Identification of a Biosynthetic Gene Cluster in Rice for Momilactones

Abstract

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 3␤-hydroxy-9␤H-pimara-7,15-dien-19,6␤-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 (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 9␤H-pimara-7,15-diene and stemar-13-ene, leading to momilactones A and B and oryzalexin S, respectively. The hypotheti...

Allelopathic momilactones A and B are implied in rice drought and salinity tolerance, not weed resistance

“…In previous research, the quantification of momilactones A and B was reported using HPLC (Lee et al 1999;Shimura et al 2007;Lee et al 2002;Kong et al 2004;Kim et al 2007;Kato-Noguchi and Ota 2013). However, their contents can also be determined by GC-MS, which have been noted in in several reports (Matsuyama 1983;Fukuta et al 2007;Kodama et al 1988), as well as in this study, using standard momilactones A and B to compare their retention times, mass spectra, and fragments.…”

Increasing Complexity of a Diterpene Synthase Reaction with a Single Residue Switch

“…Furthermore, the gene for OsKSL4 is found in the rice genome near that for the syn-CPP synthase (OsCPS4), 4 along with those for a dehydrogenase and cytochromes P450 also involved in momilactone biosynthesis. 5 Such secondary metabolism gene clusters are unusual in plants and generally are taken as an indication of strong selective pressure for production of the resulting compound operating over evolutionarily long timescales. 6 Together these findings suggest OsKSL4 has served as a syn-pimaradiene specific synthase for an extended period of time, with no selective pressure to retain catalytic features in its active site for more complex reaction mechanisms.…”

The application of synthetic biology to elucidation of plant mono-, sesqui- and diterpenoid metabolism

“…D) Both CYP99A2 & 3 can convert C19 of syn-pimaradiene to a carboxylic acid, although this olefin can be alternatively hydroxylated at C3β by CYP701A8 or C7β by CYP76M8. The C19 oxidation catalyzed by CYP99A2 & 3 is relevant to momilactone biosynthesis (Shimura et al, 2007), while those catalyzed by CYP701A8 and CYP76M8 may be relevant as well (Kitaoka et al, submitted).…”

“…Many of these genes were found to be coregulated, specifically inducible by the fungal cell wall component chitin, as well . Subsequently, genetic (RNAi) evidence was reported indicating a role for the CYPs from the chromosome 4 cluster in momilactone diterpenoid metabolism (Shimura et al, 2007), consistent with the role of the co-clustered OsCPS4 and OsKSL4 [Figure 3 ]. This information was particularly useful in enabling focused investigation of CYPs -i.e., rather than investigating all >350 CYP found in the rice genome, we then simply targeted those from the diterpenoid biosynthesis clusters, along with the few others that exhibited inducible transcription (15 total).…”

“…However, no activity was observed for the other CYPs with this approach. Knowing that at least one of the two closely related CYPs found in the cluster on chromosome 4 (i.e., CYP99A2 and 3) played a role in momilactone biosynthesis (Shimura et al, 2007), we turned to use of synthetic genes optimized for expression in E. coli, along with N-terminal modification. This led to functional expression and successful incorporation into our metabolic engineering system of at least one of these, CYP99A3, which acts on the syn-pimaradiene product of the coclustered OsCPS4 and OsKSL4, carrying out conversion of C19 from a methyl to carboxylic acid .…”