Comprehensive identification of target genes of OsTGAP1 revealed the multifaceted regulation of the gene expression responsible for the diterpenoid phytoalexin synthesis. Oryza sativa Japonica Group

Plants attacked by pathogenic microorganisms respond with a variety of defensive reaction, including the production of antimicrobial secondary metabolites known as phytoalexins. Momilactones and phytocassanes are major diterpenoid phytoalexins in rice and are synthesized from geranylgeranyl diphosphate via the upstream metabolic pathway, methylerythritol phosphate (MEP) pathway. We have previously reported that biosynthetic genes for momilactones and phytocassanes are respectively clustered on rice chromosome 4 and chromosome 2, and that an elicitor-inducible bZIP transcription factor, OsTGAP1, is involved in the regulation of the coordinated expression of biosynthetic genes for diterpenoid phytoalexins and the upstream MEP pathway genes. To clarify the cooridinated regulatory mechanism of these genes by OsTGAP1, transcriptome analysis and chromatin immunoprecipitation with next-generation sequencing (ChIP-seq) were performed identifying the OsTGAP1-target genes. We found that OsTGAP1 is involved in various processes in the regulation of diterpenoid phytoalexin production. OsDXS3, encoding a key enzyme of the MEP pathway, was the only OsTGAP1-target gene in the MEP pathway. The subsequent trans-activation assay further confirmed the OsTGAP1 direct regulation of the OsDXS3 expression. As for phytoalexin biosynthetic gene clusters, we found several OsTGAP1-binding sites at intergenic region in and near the cluster regions, suggesting that OsTGAP1 regulates the clustered phytoalexin biosynthetic genes in different manner. The presence of several transcription factors in the OsTGAP1-target genes also revealed the transcription cascade from OsTGAP1 to reach the synthesis of diterpenoid phytoalexins. These results imply the presence of multifaceted regulation by OsTGAP1 to the gene expression responsible for the inductive biosynthesis of diterpenoid phytoalexins in rice.