Plants have evolved efficient defence mechanisms to defend themselves from pathogen attack. Although many studies have focused on the transcriptional regulation of defence responses, less is known about the involvement of microRNAs (miRNAs) as post-transcriptional regulators of gene expression in plant immunity. This work investigates miRNAs that are regulated by elicitors from the blast fungus Magnaporthe oryzae in rice (Oryza sativa). Small RNA libraries were constructed from rice tissues and subjected to high-throughput sequencing for the identification of elicitor-responsive miRNAs. Target gene expression was examined by microarray analysis. Transgenic lines were used for the analysis of miRNA functioning in disease resistance. Elicitor treatment is accompanied by dynamic alterations in the expression of a significant number of miRNAs, including new members of annotated miRNAs. Novel miRNAs from rice are proposed. We report a new rice miRNA, osa-miR7695, which negatively regulates an alternatively spliced transcript of OsNramp6 (Natural resistance-associated macrophage protein 6). This novel miRNA experienced natural and domestication selection events during evolution, and its overexpression in rice confers pathogen resistance. This study highlights an miRNA-mediated regulation of OsNramp6 in disease resistance, whilst illustrating the existence of a novel regulatory network that integrates miRNA function and mRNA processing in plant immunity.

Metal ions are essential elements for all living organisms. However, metals can be toxic when present in excess. In plants, metal homeostasis is partly achieved through the function of metal transporters, including the diverse natural resistance-associated macrophage proteins (NRAMP). Among them, the OsNramp6 gene encodes a previously uncharacterized member of the rice NRAMP family that undergoes alternative splicing to produce different NRAMP6 proteins. In this work, we determined the metal transport activity and biological role of the full-length and the shortest NRAMP6 proteins (l-NRAMP6 and s-NRAMP6, respectively). Both l-NRAMP6 and s-NRAMP6 are plasma membrane-localized proteins that function as iron and manganese transporters. The expression of l-Nramp6 and s-Nramp6 is regulated during infection with the fungal pathogen Magnaporthe oryzae, albeit with different kinetics. Rice plants grown under high iron supply show stronger induction of rice defense genes and enhanced resistance to M. oryzae infection. Also, loss of function of OsNramp6 results in enhanced resistance to M. oryzae, supporting the idea that OsNramp6 negatively regulates rice immunity. Furthermore, nramp6 plants showed reduced biomass, pointing to a role of OsNramp6 in plant growth. A better understanding of OsNramp6-mediated mechanisms underlying disease resistance in rice will help in developing appropriate strategies for crop protection.

14-3-3 proteins are found in all eukaryotes where they act as regulators of diverse signalling pathways associated with a wide range of biological processes. In this study the functional characterization of the ZmGF14-6 gene encoding a maize 14-3-3 protein is reported. Gene expression analyses indicated that ZmGF14-6 is up-regulated by fungal infection and salt treatment in maize plants, whereas its expression is down-regulated by drought stress. It is reported that rice plants constitutively expressing ZmGF14-6 displayed enhanced tolerance to drought stress which was accompanied by a stronger induction of drought-associated rice genes. However, rice plants expressing ZmGF14-6 either in a constitutive or under a pathogen-inducible regime showed a higher susceptibility to infection by the fungal pathogens Fusarium verticillioides and Magnaporthe oryzae. Under infection conditions, a lower intensity in the expression of defence-related genes occurred in ZmGF14-6 rice plants. These findings support that ZmGF14-6 positively regulates drought tolerance in transgenic rice while negatively modulating the plant defence response to pathogen infection. Transient expression assays of fluorescently labelled ZmGF14-6 protein in onion epidermal cells revealed a widespread distribution of ZmGF14-6 in the cytoplasm and nucleus. Additionally, colocalization experiments of fluorescently labelled ZmGF14-6 with organelle markers, in combination with cell labelling with the endocytic tracer FM4-64, revealed a subcellular localization of ZmGF14-6 in the early endosomes. Taken together, these results improve our understanding of the role of ZmGF14-6 in stress signalling pathways, while indicating that ZmGF14-6 inversely regulates the plant response to biotic and abiotic stresses.

BackgroundMicroRNAs (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.ResultsRice 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.ConclusionsOur 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.