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

Sánchez-Sanuy F, Peris-Peris C, Tomiyama S, Okada K, Hsing YI, San Segundo B, Campo S.

BMC Plant Biol. 2019 Dec 18;19(1):563. doi: 10.1186/s12870-019-2156-5Abstract

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.

See https://bmcplantbiol.biomedcentral.com/articles/10.1186/s12870-019-2156-5

Figure 2: Resistance of MIR7695-Ac mutant plants to M. oryzae infection. a RT-qPCR analysis of MIR7695 precursor transcripts (left panel) and miR7695 target (Nramp6.8, Os01g0503400.8) in homozygous mutant (MIR7695-Ac) and WT (segregated azygous, WT-Az) plants. Data are mean ± SE (n = 3) (Student t test, *p < 0.05). Lower panel: northern blot analysis of mature miR7695 using the miR7695.3-3p sequence as the hybridization probe (Additional file 2: Table S1). As a loading control, the RNA blot was stained with ethidium bromide (EtBr) (b) Experimental validation of miR7695-mediated cleavage of OsNramp6.8 transcripts by 5′-RLM-RACE. Schematic representation of the OsNramp6.8 (upper panel), showing the coding sequence (blue), 5’UTR (green), and 3’UTR (pink). Boxes, exons; lines, introns. Gene-specific primers were used for 5′-RACE and the resulting PCR products were sequenced. The identified cleavage site is indicated by an arrow and the number above indicate the detected cleavage site of independent clones. c Leaves of 3-week-old plants were sprayed with a M. oryzae spore suspension. The second leaf was photographed at 7 days post-inoculation. d Percentage of leaf area affected by blast lesions (upper panel). Relative fungal biomass (lower panel) was determined by qPCR as the ratio of M. oryzae 28S ribosomal DNA to the rice Ubiquitin1 gene (primers in Additional file 2: Table S1). Data are mean ± SE (n = 7) from 1 experiment (Student t test, *p < 0.05). Four independent infection assays were performed with similar results. e RT-qPCR analysis of OsPR1a transcripts at different times after inoculation with M. oryzae spores. Blast infection was carried out as in (c). Data are mean ± SE (n = 3, each biological replicate is a pool of 3 individual leaves) (Student t test, **p < 0.01 ***p < 0.001; infected vs non-infected). Mock inoculated (control) plants; +, M. oryzae-infected plants.