Abstract
Phytoalexins are specialised antimicrobial metabolites that are produced by plants in response to pathogen attack. Momilactones and phytocassanes are the major diterpenoid phytoalexins in rice and are synthesised from geranylgeranyl diphosphate, which is derived from the methylerythritol phosphate (MEP) pathway. The hyperaccumulation of momilactones and phytocassanes due to the hyperinductive expression of the relevant biosynthetic genes and the MEP pathway gene OsDXS3 in OsTGAP1-overexpressing (OsTGAP1ox) rice cells has previously been shown to be stimulated by the chitin oligosaccharide elicitor. In this study, to clarify the mechanisms of the elicitor-stimulated coordinated hyperinduction of these phytoalexin biosynthetic genes in OsTGAP1ox cells, transcriptome analysis and chromatin immunoprecipitation with next-generation sequencing were performed, resulting in the identification of 122 OsTGAP1 target genes. Transcriptome analysis revealed that nearly all of the momilactone and phytocassane biosynthetic genes, which are clustered on chromosomes 4 and 2, respectively, and the MEP pathway genes were hyperinductively expressed in the elicitor-stimulated OsTGAP1ox cells. Unexpectedly, none of the clustered genes was included among the OsTGAP1 target genes, suggesting that OsTGAP1 did not directly regulate the expression of these biosynthetic genes through binding to each promoter region. Interestingly, however, several OsTGAP1-binding regions were found in the intergenic regions among and near the cluster regions. Concerning the MEP pathway genes, only OsDXS3, which encodes a key enzyme of the MEP pathway, possessed an OsTGAP1-binding region in its upstream region. A subsequent transactivation assay further confirmed the direct regulation of OsDXS3 expression by OsTGAP1, but other MEP pathway genes were not included among the OsTGAP1 target genes. Collectively, these results suggest that OsTGAP1 participates in the enhanced accumulation of diterpenoid phytoalexins, primarily through mechanisms other than the direct transcriptional regulation of the genes involved in the biosynthetic pathway of these phytoalexins.
Highlights
Phytoalexins are specialised antimicrobial metabolites that are produced by plants in response to pathogen attack [1]
Concerning the methylerythritol phosphate (MEP) pathway genes, it was found that only OsDXS3 possessed an OsTGAP1-binding region in its upstream region and that OsTGAP1 directly regulated OsDXS3 expression, while other MEP pathway genes were not included among the OsTGAP1 target genes
When either TGACGT sequence in this region was mutated (m1 and m2), the binding of glutathione S-transferase (GST)-OsTGAP1 to the DNA probe was weakened. When both TGACGT sequences were mutated (m3), GST-OsTGAP1 no longer bound to the DNA probe (Figure 5B). These results indicate that OsTGAP1 binds to the OsDXS3 promoter via two TGACGT sequences
Summary
Phytoalexins are specialised antimicrobial metabolites that are produced by plants in response to pathogen attack [1]. In suspension-cultured rice cells, the MEP pathway genes exhibit elicitor-induced expression, and the MEP pathway is required for the production of sufficient amounts of diterpenoid phytoalexins [7]. Four P450 monooxygenases (CYP71Z7, CYP76M7, CYP76M8, and CYP701A8/OsKOL4) are involved in the oxidation of ent-cassa12,15-diene [13,14,15,16]. The momilactone and phytocassane biosynthetic genes are localized in narrow regions of chromosomes 4 and 2, respectively, creating functional gene clusters [11,13]. These biosynthetic genes exhibit the temporally coordinated expression of mRNAs after treatment with a biotic elicitor in suspensioncultured rice cells [7,11]
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
