Osa-miR7695 enhances transcriptional priming in defense responses against the rice blast fungus.

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.

OsDCL1a activation impairs phytoalexin biosynthesis and compromises disease resistance in rice.

Background and Aims: MicroRNAs (miRNAs) are small non-coding RNAs that act as post-transcriptional regulators of gene expression via sequence-specific cleavage or translational repression of target transcripts. They are transcribed as long single-stranded RNA precursors with unique stem-loop structures that are processed by a DICER-Like (DCL) ribonuclease, typically DCL1, to produce mature miRNAs. Although a plethora of miRNAs have been found to be regulated by pathogen infection in plants, the biological function of most miRNAs remains largely unknown. Here, the contribution of OsDCL1 to rice immunity was investigated. Methods: Activation-tagged Osdcl1a (Osdcl1a-Ac) rice mutants were examined for resistance to pathogen infection. mRNA and small RNA deep sequencing, quantitative real-time PCR (RT-qPCR) and stem-loop reverse tanscripion-PCR (RT-PCR) were used to examine DCL1a-mediated alterations in the rice transcriptome. Rice diterpene phytoalexins were quantified by liquid chromatography-tandem mass spectrometry (LC-MSMS). Accumulation of O2·- was determined by nitroblue tetrazolium (NBT) staining. Key Results: dcl1a-Ac mutants exhibit enhanced susceptibility to infection by fungal pathogens which was associated with a weaker induction of defence gene expression. Comparison of the mRNA and miRNA transcriptomes of dcl1a-Ac and wild-type plants revealed misregulation of genes involved in detoxification of reactive oxygen species. Consequently, dcl1a-Ac plants accumulated O2·- in their leaves and were more sensitive to methyl viologen-induced oxidative stress. Furthermore, dcl1a-Ac plants showed downregulation of diterpenoid phytoalexin biosynthetic genes, these genes also being weakly induced during pathogen infection. Upon pathogen challenge, dcl1a-Ac plants failed to accumulate major diterpenoid phytoalexins. OsDCL1a activation resulted in marked alterations in the rice miRNAome, including both upregulation and downregulation of miRNAs. Conclusions: OsDCL1a activation enhances susceptibility to infection by fungal pathogens in rice. Activation of OsDCL1a represses the pathogen-inducible host defence response and negatively regulates diterpenoid phytoalexin production. These findings provide a basis to understand the molecular mechanisms through which OsDCL1a mediates rice immunity.

Characterization of diterpene synthase genes in the wild rice species Oryza brachyatha provides evolutionary insight into rice phytoalexin biosynthesis.

Cultivated rice (Oryza sativa; Os) produces a variety of labdane-related diterpenoids; not only phytohormone gibberellins (GAs) but also phytoalexins for defense including phytocassanes, momilactones and oryzalexins. Their carbon skeleton diterpenes are constructed from geranylgeranyl diphosphate via ent-copalyl diphosphate (ent-CDP) or its diastereomer syn-CDP. These two-step reactions are successively catalyzed by homologs of the two diterpene synthases, ent-CDP synthase (ent-CPS) and ent-kaurene synthase (KS) that are responsible for the biosynthesis of GAs; e.g. OsCPS4 and OsKSL8 that are involved in the biosynthesis of oryzalexin S, a rice phytoalexin. Oryza brachyantha (Ob) is the most distant wild rice species from Os among the Oryza genus. We previously reported that the Ob genome contains ObCPS_11g, ObKSL8-a, ObKSL8-b and ObKSL8-c for specialized metabolism at a locus similar to the OsKSL8 locus on chromosome 11. These Ob genes are closely related to OsCPS4 and OsKSL8, respectively. We herein characterize the diterpene synthase genes in Ob, using functional analyses and expression analysis. Recombinant OsKSL8 and ObKSL8-a showed the same in vitro function when syn-CDP or normal-CDP were used as substrates. Nonetheless, our results suggest that Ob produces normal-CDP-related diterpenoid phytoalexins, presumably via ObKSL8-a, while Os produces a syn-CDP-related phytoalexin, oryzalexin S, via OsKSL8. This difference must be due to the kinds of CPS that are present in each species; Os has OsCPS4 encoding syn-CPS, while Ob has ObCPS_11g encoding normal-CPS. Thus, we propose the evolutionary history underlying oryzalexin S biosynthesis: the gain of a syn-CPS was a critical event allowing the biosynthesis of oryzalexin S.