Momilactones are bioactive diterpenoids that contribute to plant defense against pathogens and allelopathic interactions between plants. Both cultivated and wild grass species ofOryzaandEchinochloa crus-galli(barnyard grass) produce momilactones using a biosynthetic gene cluster (BGC) in their genomes. The bryophyteCalohypnum plumiforme(formerlyHypnum plumaeforme) also produces momilactones, and the bifunctional diterpene cyclase geneCpDTC1/HpDTC1,which is responsible for the production of the diterpene framework, has been characterized. To understand the molecular architecture of the momilactone biosynthetic genes in the moss genome and their evolutionary relationships with other momilactone-producing plants, we sequenced and annotated theC. plumiformegenome. The data revealed a 150-kb genomic region that contains two cytochrome P450 genes, theCpDTC1/HpDTC1gene and the “dehydrogenase momilactone A synthase” gene tandemly arranged and inductively transcribed following stress exposure. The predicted enzymatic functions in yeast and recombinant assay and the successful pathway reconstitution inNicotiana benthamianasuggest that it is a functional BGC responsible for momilactone production. Furthermore, in a survey of genomic sequences of a broad range of plant species, we found that momilactone BGC is limited to the two grasses (OryzaandEchinochloa) andC. plumiforme, with no synteny among these genomes. These results indicate that while the gene cluster inC. plumiformeis functionally similar to that in rice and barnyard grass, it is likely a product of convergent evolution. To the best of our knowledge, this report of a BGC for a specialized plant defense metabolite in bryophytes is unique.

Biosynthesis of sakuranetin, a flavonoid anti-fungal phytoalexin that occurs in rice, is highly dependent on jasmonic acid (JA) signalling and induced by a variety of environmental stimuli. We previously identified OsNOMT, which encodes naringenin 7-O-methyltransferase (NOMT); NOMT is a key enzyme for sakuranetin production. Although OsNOMT expression is induced by JA treatment, the regulation mechanism that activates the biosynthetic pathway of sakuranetin has not yet been elucidated. In this study, we show that JA-inducible basic helix-loop-helix transcriptional factor OsMYC2 drastically enhances the activity of the OsNOMT promoter and is essential for JA-inducible sakuranetin production. In addition, we identified 2 collaborators of OsMYC2, OsMYC2-like protein 1 and 2 (OsMYL1 and OsMYL2) that further activated the OsNOMT promoter in synergy with OsMYC2. Physical interaction of OsMYC2 with OsMYL1 and OsMYL2 further supported the idea that these interactions lead to the enhancement of the transactivation activity of OsMYC2. Our results indicate that JA signalling via OsMYC2 is reinforced by OsMYL1 and OsMYL2, resulting in the inductive production of sakuranetin during defence responses in rice.

Background: MicroRNAs (miRNAs) are small non-coding RNAs that regulate gene expression at the post-transcriptional level in eukaryotes. In rice, MIR7695 expression is regulated by infection with the rice blast fungus Magnaporthe oryzae with subsequent down-regulation of an alternatively spliced transcript of natural resistance-associated macrophage protein 6 (OsNramp6). NRAMP6 functions as an iron transporter in rice. Results: Rice plants grown under high iron supply showed blast resistance, which supports that iron is a factor in controlling blast resistance. During pathogen infection, iron accumulated in the vicinity of M. oryzae appressoria, the sites of pathogen entry, and in cells surrounding infected regions of the rice leaf. Activation-tagged MIR7695 rice plants (MIR7695-Ac) exhibited enhanced iron accumulation and resistance to M. oryzae infection. RNA-seq analysis revealed that blast resistance in MIR7695-Ac plants was associated with strong induction of defense-related genes, including pathogenesis-related and diterpenoid biosynthetic genes. Levels of phytoalexins during pathogen infection were higher in MIR7695-Ac than wild-type plants. Early phytoalexin biosynthetic genes, OsCPS2 and OsCPS4, were also highly upregulated in wild-type rice plants grown under high iron supply. Conclusions: Our data support a positive role of miR7695 in regulating rice immunity that further underpin links between defense and iron signaling in rice. These findings provides a basis to better understand regulatory mechanisms involved in rice immunity in which miR7695 participates which has a great potential for the development of strategies to improve blast resistance in rice.