Osa-miR169 Negatively Regulates Rice Immunity against the Blast Fungus Magnaporthe oryzae

Abstract

miR169 is a conserved microRNA (miRNA) family involved in plant development and stress-induced responses. However, how miR169 functions in rice immunity remains unclear. Here, we show that miR169 acts as a negative regulator in rice immunity against the blast fungus Magnaporthe oryzae by repressing the expression of nuclear factor Y-A (NF-YA) genes. The accumulation of miR169 was significantly increased in a susceptible accession but slightly fluctuated in a resistant accession upon M. oryzae infection. Consistently, the transgenic lines overexpressing miR169a became hyper-susceptible to different M. oryzae strains associated with reduced expression of defense-related genes and lack of hydrogen peroxide accumulation at the infection site. Consequently, the expression of its target genes, the NF-YA family members, was down-regulated by the overexpression of miR169a at either transcriptional or translational level. On the contrary, overexpression of a target mimicry that acts as a sponge to trap miR169a led to enhanced resistance to M. oryzae. In addition, three of miR169's target genes were also differentially up-regulated in the resistant accession upon M. oryzae infection. Taken together, our data indicate that miR169 negatively regulates rice immunity against M. oryzae by differentially repressing its target genes and provide the potential to engineer rice blast resistance via a miRNA.

Keywords: miR169; microRNA; nuclear factor Y-A; rice blast; target mimicry.

FIGURE 1

Differential accumulation of miR169 in susceptible and resistant accessions upon Magnaporthe oryzae infection. (A) Representative leaf sections from the susceptible accession LTH and the resistant accession IRBL9-W show the blast disease phenotype. Three-leaf-stage seedlings were inoculated with Guy11 spore suspensions, and disease phenotypes were recorded at 5 dpi. Similar results were obtained in at least two independent experiments. (B,C) Differential accumulation of different miR169 isoforms in the indicated accessions upon M. oryzae infection. RNA was extracted at the indicated time points for qRT-PCR analysis. The transcriptional level of miR169 family members was normalized to miR169a in untreated LTH (0 h). SnRNA U6 served as an internal reference. (D) The sum accumulation of total miR169 in the indicated accessions upon M. oryzae infection. Values in (B–D) are means of three replications. Error bars indicate SD. The letters above the bars indicate significant differences (P < 0.01) as determined by a one-way ANOVA followed by post hoc Tukey HSD analysis. Similar results were obtained in at least two independent experiments.

FIGURE 2

Overexpressing miR169a results in enhanced susceptibility to M. oryzae. (A) Comparison of miR169a accumulation in the transgenic lines harboring 35S:miR169a. SnRNA U6 served as the internal reference. The accumulation level of miR169a was normalized to that of the control plants. Values are means of three replications. Error bars indicate SD. The letters above the bars indicate significant differences (P < 0.01) as determined by a one-way ANOVA followed by post hoc Tukey HSD analysis. Similar results were obtained in at least two independent experiments. Comparison of disease phenotypes and composition of disease lesion types caused by the M. oryzae strain Y34 (B), GZ8 (C) and Guy11 (D), respectively. Lesions in 30 diseased leaves were analyzed at 5 dpi. Similar results were obtained in at least two independent experiments.

FIGURE 3

Overexpressing miR169a results in compromised defense responses. (A–C) Expression pattern of the indicated defense-related genes in the transgenic lines harboring 35S:miR169a and the control plants, respectively, upon M. oryzae infection. RNA was extracted at the indicated time points for qRT-PCR analysis. The indicated mRNA level was normalized to that in the untreated control plants (0 h). Values are means of three replications. Error bars indicate SD. The letters above the bars indicate significant differences (P < 0.01) as determined by a one-way ANOVA followed by post hoc Tukey HSD analysis. Similar results were obtained in at least two independent experiments. (D) Representative leaf sections from the indicated lines show H₂O₂ accumulation at 2 dpi, respectively. Note that there was no or trace amounts of H₂O₂ accumulation in the transgenic lines overexpressing miR169a, but high level of H₂O₂ was observed around the appressorium (arrow) in the leaf cell from the control plants. The fungal structure and accumulated H₂O₂ were stained by Trypan blue and DAB, respectively. Bars = 10 μm. Similar results were obtained in at least two independent experiments.

FIGURE 4

Overexpressing miR169a leads to increased M. oryzae growth. (A) Representative Laser Scanning Confocal Microscopy (LSCM) images show the growth of the M. oryzae strain GZ8 at 12 hours post inoculation (hpi) and 36 hpi on sheath cells of the indicated transgenic lines harboring 35S:miR169a and the control plants, respectively. Note that the appressoria (arrows) were formed at 12 hpi on miR169a-overexpression plants but delayed on control plants. Invasive hyphae were formed at 36 hpi on both control and miR169a-overexpressing plants, but only extended into the neighbor cells (arrowheads) of miR169a-overexpressing plants. Bars = 20 μm. (B) The quantitative analysis of M. oryzae growth. More than 200 conidia in each lines were analyzed. Similar results were obtained in at least two independent experiments.

FIGURE 5

MiR169a represses the expression of its target genes at transcriptional or translational level. (A) Comparison of the relative mRNA levels of the indicated miR169’s target genes in the indicated lines. RNA was extracted in the indicated lines for qRT-PCR analysis. The mRNA level was normalized to that in the control plants. Values are means of three replications. Error bars indicate SD. The letters above the bars indicate significant differences (P < 0.01) as determined by a one-way ANOVA followed by post hoc Tukey HSD analysis. Western blotting analysis (B) and confocal images (C,D) show that miR169a suppressed the protein accumulation of Os03g29760 but did not affect the protein level of YFP control. The indicated YFP-3′-UTR_Os03g29760 and YFP-based reporter constructs were transiently expressed alone or co-expressed with miR169a (C) or/and the miR169 target mimicry MIM169a (D) in Nicotiana benthamiana leaves using Agrobacterium-mediated infiltration at the indicated optical density (O.D.) concentration. Protein extracts from the same amount of infiltrated leaves were subjected to Western blot analysis using anti-GFP sera. The Coomassie Blue stained Rubisco indicated the equal sample loading (B). The alignments of miR169a with Os03g29760 target sequence (C) and MIM169a with miR169a (D) were listed below the images, respectively. Size bars, 40 μm. Similar results were obtained in at least two independent experiments.

FIGURE 6

Expression of the miR169a target mimicry results in enhanced resistance to M. oryzae. (A) Comparison of the transcriptional level of miR169’s target genes in control plants and the transgenic lines expressing the miR169a target mimicry MIM169a. RNA was extracted in indicated lines for qRT-PCR analysis. mRNA level was normalized to that in control plants. Values are means of three replications. Error bars indicate SD. The letters above the bars indicate significant differences (P < 0.01) as determined by a one-way ANOVA followed by post hoc Tukey HSD analysis. (B) MIM169a transgenic lines display enhanced resistance to the M. oryzae strain GZ8. The relative fungal mass on the inoculated leaves of the indicated transgenic lines was measured by using the ratio of DNA level of M. oryzae Pot2 against the rice genomic ubiquitin DNA level. Values are means of three replications. Error bars indicate SD. The letters above the bars indicate significant differences (P < 0.01) as determined by a one-way ANOVA followed by post hoc Tukey HSD analysis. Similar results were obtained in at least two independent experiments.

FIGURE 7

Differential expression of miR169’s target genes in susceptible and resistant accessions upon M. oryzae infection. (A–C) Expression of the indicated target genes in LTH and IRBL9-W upon M. oryzae infection. RNA was extracted at the indicated time points for quantitative RT-PCR analysis. mRNA level was normalized to that in untreated samples (0 h). Values are means of three replications. Error bars indicate SD. The letters above the bars indicate significant differences (P < 0.01) as determined by a one-way ANOVA followed by post hoc Tukey HSD analysis. Similar results were obtained in at least two independent experiments.