Fig. 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.