Elucidation of mechanisms regulating expression of a phytoalexin biosynthetic gene cluster in rice

我们发现两个P450基因(CYP99A2和CYP99A3)和一个可能的脱氢酶基因(AK103462)与两个二萜环化酶基因一起形成了一个位于水稻基因组4号染色体上的基因簇，这两个基因参与了吗内酯的生物合成。此外，在悬浮培养的水稻细胞中，甲壳素低聚糖激发子处理后，5个聚集的基因被协同诱导，这表明甜蜜内酯生物合成基因聚集在4号染色体上。在上述背景的基础上，本研究旨在证明甜蜜素生物合成基因簇的存在，并阐明该基因簇协调表达的调控机制。我们发现AK103462编码木霉内酯A合成酶，并且CYP99A2和CYP99A3的双重敲除特异性地抑制了激发子诱导的木霉内酯的产生。因此，我们证明在水稻的4号染色体上存在一个由4个或5个基因组成的动内酯生物合成基因簇。此外，我们还鉴定了一个激发子诱导的碱性亮氨酸拉链(BZIP)转录因子OsTGAP1，它协调调节丁香内酯生物合成基因簇中所有五个基因的表达。我们还发现OsTGAP1可以影响植物生物合成基因和上游甲基赤藓糖醇磷酸(MEP)途径基因的表达。因此，我们认为OsTGAP1在水稻诱导二萜类植物保卫素产生相关基因的协调转录中起关键作用。这些研究为我们阐明水稻二萜类植物保卫素产生的调控机制提供了重要的基础信息。

We found that two P450 genes (CYP99A2 and CYP99A3) and a putative dehydrogenase gene (AK103462) forms a gene cluster on chromosome 4 of the rice genome together with two diterpene cyclase genes that are involved in biosynthesis of momilactones. In addition, the five clustered genes were coordinately induced after treatment with a chitin oligosaccharide elicitor in suspension-cultured rice cells, suggesting that the momilactone biosynthesis genes are clustered on chromosome 4.On the basis of the above background, this project aimed to demonstrate the presence of a momilactone biosynthetic gene cluster and elucidate regulatory mechanisms for coordinate expression of the gene cluster. We indicated that AK103462 encodes momilactone A synthase, and that the double knockdown of CYP99A2 and CYP99A3 specifically suppressed the elicitor inducible production of momilactones. We thus demonstrated that a momilactone biosynthetic gene cluster consisting of four or five genes is present on chromosome 4 in rice. In addition, we identified an elicitor-inducible basic leucine zipper (bZIP) transcription factor, OsTGAP1, which coordinately regulates the expression of all five genes in the momilactone biosynthetic gene cluster. We also showed that OsTGAP1 can influence the expression of a phytocassane biosynthetic gene and an upstream methylerythritol phosphate (MEP) pathway gene. We thus indicated that OsTGAP1 functions as a key regulator of the coordinated transcription of genes involved in inductive diterpenoid phytoalexins production in rice.All the studies presented here give us important basic information to clarify regulatory mechanisms for production of diterpenoid phytoalexins in rice.