Inducible Enrichment of Osa-miR1432 Confers Rice Bacterial Blight Resistance through Suppressing OsCaML2

Abstract

MicroRNAs (miRNAs) handle immune response to pathogens by adjusting the function of target genes in plants. However, the experimentally documented miRNA/target modules implicated in the interplay between rice and Xanthomonas oryzae pv. oryzae (Xoo) are still in the early stages. Herein, the expression of osa-miR1432 was induced in resistant genotype IRBB5, but not susceptible genotype IR24, under Xoo strain PXO86 attack. Overexpressed osa-miR1432 heightened rice disease resistance to Xoo, indicated by enhancive enrichment of defense marker genes, raised reactive oxygen species (ROS) levels, repressed bacterial growth and shortened leaf lesion length, whilst the disruptive accumulation of osa-miR1432 accelerated rice susceptibility to Xoo infection. Noticeably, OsCaML2 (LOC_Os03g59770) was experimentally confirmed as a target gene of osa-miR1432, and the overexpressing OsCaML2 transgenic plants exhibited compromised resistance to Xoo infestation. Our results indicate that osa-miR1432 and OsCaML2 were differently responsive to Xoo invasion at the transcriptional level and fine-tune rice resistance to Xoo infection, which may be referable in resistance gene discovery and valuable in the pursuit of improving Xoo resistance in rice breeding.

Keywords: OsCaML2; osa-miR1432; rice bacterial blight.

Figure 1

Differential expression patterns of osa-miR1432 in susceptible and resistant genotypes upon Xoo infection. (A) Phenotype of representative leaf sections from susceptible genotype IR24 and resistant genotype IRBB5 at 20 dpi with Xoo strain PXO86. (B) Accumulation of osa-miR1432 was monitored using RT-qPCR in susceptible and resistant genotypes under Xoo strain PXO86 and mock treatment. Scale bar, 2 cm. Error bars indicate standard deviation (n = 3). Different letters above the bars indicate significant differences (p < 0.01) determined by one-way analysis of variance (ANOVA) followed by post hoc Tukey HSD analysis.

Figure 2

Osa-miR1432 modulates Xoo resistance in rice. (A) The expression detection of osa-miR1432 in representative overexpressing transgenic lines. SnRNA U6 served as the internal reference. (B) Lesions on OE1432 lines (OE1432-1 and OE1432-7) and the TP309 at 20 dpi. Scale bar, 2 cm. (C) Lesion lengths on OE1432 plants (n = 20) at 20 dpi. (D) Bacterial growth indicated by the numbers of colony-forming units (cfu) per leaf in OE1432 lines and TP309. (E) The basic expression of OsPBZ1, OsPR1a and OsPR1b in OE1432 lines and TP309. (F) The histochemical detection of ROS levels using NBT and DAB staining in OE1432 lines and TP309 at 4 dpi. Error bars indicate standard deviation (n = 3). Scale bar, 2 mm. Different letters above the bars indicate significant differences (p < 0.01) determined by one-way analysis of variance (ANOVA) followed by post hoc Tukey HSD analysis.

Figure 3

Restraining the expressing of osa-miR1432 results in susceptibility to Xoo attack in rice. (A) The expression detection of osa-miR1432 in MIM1432 lines, SnRNA U6, served as the internal reference. (B) Lesions on MIM1432 lines (MIM1432-3 and MIM1432-8) and TP309 at 20 dpi. Scale bar, 2 cm. (C) Lesion lengths on MIM1432 lines (n = 20) at 20 dpi. (D) Bacterial growth as indicated by the numbers of colony-forming units (cfu) per leaf in MIM1432 lines and TP309. (E) The basic expression of OsPBZ1, OsPR1a and OsPR1b in MIM1432 transgenic lines and TP309. (F) The histochemical detection of ROS levels using NBT and DAB staining in MIM1432 lines and TP309 at 4 dpi. Error bars indicate standard deviation (n = 3). Scale bar, 2 mm. Different letters above the bars indicate significant differences (p < 0.01) determined by one-way analysis of variance (ANOVA) followed by post hoc Tukey HSD analysis.

Figure 4

The candidate targets of osa-miR1432 have differential expression profiles in susceptible and resistant genotypes upon Xoo infection. (A) The relative expression levels of the candidate targets in the OE1432 and MIM1432 lines. (B–D) The expression pattern of OsCaML2, OsCAS and OsZIP6 in susceptible and resistant genotypes upon Xoo or mock treatment. Error bars indicate standard deviation (n = 3). Different letters above the bars indicate significant differences (p < 0.01) determined by one-way analysis of variance (ANOVA) followed by post hoc Tukey HSD analysis.

Figure 5

OsCaML2 negatively regulates disease resistance against Xoo in rice. (A) Accumulation of OsCaML2 in the OEOsCaML2 transgenic lines. (B) Lesions on respective OEOsCaML2 lines (OEOsCaML2-5 and OEOsCaML2-6) and the TP309 at 20 dpi. Scale bar, 2 cm. (C) Lesion lengths on OEOsCaML2 plants (n = 20) at 20 dpi. (D) Bacterial growth as indicated by the numbers of colony-forming units (cfu) per leaf for OEOsCaML2 lines and the TP309. (E) Expression pattern of OsPBZ1, OsPR1a and OsPR1b in OEOsCaML2 transgenic lines and the TP309. (F) The histochemical detection of ROS levels using NBT and DAB staining in OEOsCaML2 transgenic lines and the TP309 after Xoo infection. Error bars indicate SD (n = 3). Scale bar, 2 mm. Different letters above the bars indicate significant differences (p < 0.01) determined by one-way analysis of variance (ANOVA) followed by post hoc Tukey HSD analysis.