miR396- OsGRF s Module Balances Growth and Rice Blast Disease-Resistance

Abstract

Fitness cost is a common phenomenon in rice blast disease-resistance breeding. MiR396 is a highly conserved microRNA (miRNA) family targeting Growth Regulating Factor (OsGRF) genes. Mutation at the target site of miR396 in certain OsGRF gene or blocking miR396 expression leads to increased grain yield. Here we demonstrated that fitness cost can be trade-off in miR396-OsGRFs module via balancing growth and immunity against the blast fungus. The accumulation of miR396 isoforms was significantly increased in a susceptible accession, but fluctuated in a resistant accession upon infection of Magnaporthe oryzae. The transgenic lines over-expressing different miR396 isoforms were highly susceptible to M. oryzae. In contrast, overexpressing target mimicry of miR396 to block its function led to enhanced resistance to M. oryzae in addition to improved yield traits. Moreover, transgenic plants overexpressing OsGRF6, OsGRF7, OsGRF8, and OsGRF9 exhibited enhanced resistance to M. oryzae, but showed different alteration of growth. While overexpression of OsGRF7 led to defects in growth, overexpression of OsGRF6, OsGRF8, and OsGRF9 resulted in better or no significant change of yield traits. Collectively, our results indicate that miR396 negatively regulates rice blast disease- resistance via suppressing multiple OsGRFs, which in turn differentially control growth and yield. Therefore, miR396-OsGRFs could be a potential module to demolish fitness cost in rice blast disease-resistance breeding.

Keywords: Oryza sativa; OsGRF; disease resistance; fitness cost; miR396; rice blast disease.

FIGURE 1

Magnaporthe oryzae infection induces differential accumulation of miR396 isoforms in susceptible and resistant rice accessions. (A) Representative leaf section of LTH (susceptible) and IRBLkm-Ts (resistant) showing the blast disease phenotype. (B) Accumulation of the indicated miR396 isoforms in LTH and IRBLkm-Ts upon M. oryzae (or mock inoculated) infection. RNA samples were extracted from leaves collected at different time points and the expression of the indicated miR396 isoforms were performed using Stem-loop RT-qPCR. SnRNA U6 served as the internal reference. Error bars indicate SD from three technical replicates. The letters above the bars indicate significant differences at a P-value < 0.01, as determined by a one-way ANOVA followed by post hoc Tukey HSD analysis. The experiment was repeated twice with similar results.

FIGURE 2

Overexpression of four miR396 isoforms resulted in enhanced susceptibility to M. oryzae. (A) Accumulation of the indicated four miR396 isoforms in the respective transgenic lines. (B) Reduction of OsGRF genes in the indicated transgenic lines. (C) Blast disease assay on the indicated lines. Punch inoculation of M. oryzae strain Zhong1 on 4–5 week old leaves from wild type EV (NPB) and the indicated transgenic lines overexpressing four miR398 isoforms. Disease severity was recorded and evaluated at 5 days post inoculation. Relative fungal biomass is determined by examining the expression level of M. oryzae Pot2 gene against OsUbiquitin DNA level. (D) Representative epi-fluorescent images of sheath cells from EV and the indicated transgenic lines infected by eGFP-tagged blast isolate GZ8. Bars = 100 μm. (E) Quantification analysis on the progress of fungal infection at 12, 24, and 48 hpi. Around 200 conidia in each line were analyzed. Similar results were obtained in at least two independent experiments. (F) DAB staining shows H₂O₂ accumulation at the infection sites of the indicated lines at 2 days post inoculation (dpi). Bars = 100 μm. Arrows indicate appressoria. In (A–C), error bars indicate SD (n = 3). The letters above the bars indicate significant differences at a P-value < 0.01, as determined by a one-way ANOVA followed by post hoc Tukey HSD analysis. The asterisk above the bars indicate significant differences between EV and the indicated transgenic lines at a P-value < 0.01, as determined by Student’s t-test.

FIGURE 3

MiR396 represses the expression of its target genes at translational level. (A) Western blotting analysis and (B,C) confocal images (bars = 25 μm) show that miR396d suppressed the accumulation of GRF6_ts-YFP but did not affect the protein level of YFP control. The indicated 35S:GRF6_ts-YFP and YFP-based reporter constructs were transiently expressed alone or co-expressed with miR396d (B) or/and the miR396 target mimicry MIM396d (C) 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 Ponceau S stained Rubisco served as loading control (A). The alignments of miR396d with OsGRF6 target sequence (B) and MIM396d with miR396d (C) were listed below the images, respectively.

FIGURE 4

Preferential targeting of miR396 isoforms against OsGRFs. (A) Multiple alignment of miR396d target sites in OsGRF’s. The target genes were classified into four categories (Reporter 1, Reporter 2, Reporter 3, and Reporter 4) based on sequence homology of target site. (B) Confocal images show that miR396 isoforms differentially suppresses the protein accumulation of different OsGRF’s reporter genes. The indicated YFP-based reporter constructs were transiently expressed alone or co-expressed with miR396 isoforms in Nicotiana benthamiana. Bars = 25 μm. (C) RNA samples were collected from infiltrated leaves and the transcriptional regulation of OsGRF’s homologs by miR396 isoforms were studied using RT-qPCR. Error bars indicate SD from three technical replicates. The letters above the bars indicate significant differences at a P-value < 0.01, as determined by a one-way ANOVA followed by post hoc Tukey HSD analysis. This experiment was repeated at least three times with similar results.

FIGURE 5

Overexpression of miR396 target mimicry results in enhanced resistance to M. oryzae with improved yield traits. RT-qPCR analyses show the expression of miR396 isoforms (A) and its target genes (B) in control plants (EV, empty vector in YB background) and transgenic lines over-expressing target mimicry of miR396. The accumulation level was normalized to that of the control plants. (C) Punch inoculation of 4–5 week old leaves from EV (YB), MIM396-1 and MIM396-2 rice plants show disease severity of M. oryzae (Zhong1, 1 × 10⁵ spore/ml conc.) at 5 day post inoculation. Relative fungal biomass is determined by examining the expression level of M. oryzae Pot2 gene against OsUbiquitin DNA level. (D) Representative confocal images of sheath cells from EV, MIM396-1 and MIM396-2 infected by eGFP-tagged blast isolate GZ8. Bars = 20 μm. (E) Quantification analysis on the progress of fungal infection at 24, 36, and 48 hpi. Around 200 conidia in each line were analyzed. Similar results were obtained in at least two independent experiments. (F) DAB staining shows H₂O₂ accumulation at the infection sites of the indicated lines at 2 days post inoculation (dpi). Arrows indicate appressoria. Bars = 100 μm. (G–I) Comparison of gross plant (G), scale bar = 14 cm; and panicle morphologies (H), scale bar = 5 cm; and seed length and seed width (I), scale bar = 10 mm, of EV (YB) and miR396 mimic (MIM396-1 and MIM396-2) plants. In (A–C), error bars indicate SD from three technical replicates. The letters above the bars indicate significant differences at a P-value < 0.01, as determined by a one-way ANOVA followed by post hoc Tukey HSD analysis.

FIGURE 6

Differential expression of miR396’s target genes in susceptible and resistant accessions upon M. oryzae infection. (A–D) RT-qPCR analyses show the expression of the indicated OsGRF genes in LTH and IRBLkm-Ts at the indicated time points upon M. oryzae infection. Relative mRNA level was normalized to that in mock samples before inoculation (0h). Values are means of three technical replicates. 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.

FIGURE 7

Function of four OsGRF genes in rice blast resistance. (A) RT-qPCR analyses show the relative expression of OsGRF6, OsGRF7, OsGRF8, and OsGRF9 in the indicated transgenic lines. OsUbiquitin served as the internal reference. The accumulation level was normalized to that of the control plants (EV). (B) Disease severity of the indicated lines at 5 days post inoculation of M. oryzae strain Zhong1. Punch inoculation was conducted on 4–5 week old leaves of wild type (EV) and the indicated transgenic lines. Relative fungal biomass is determined by examining the expression level of M. oryzae Pot2 gene against OsUbiquitin DNA level. (C) Representative confocal images of sheath cells from the indicated lines infected by eGFP-tagged blast isolate GZ8. Bars = 20 μm. (D) Quantification analysis on the progress of fungal infection at 24 and 36 hpi. Around 200 conidia in each line were analyzed. Similar results were obtained in at least two independent experiments. In (A,B), error bars indicate SD (n = 3). The asterisk above the bars indicate significant differences between EV and the indicated transgenic lines at a P-value < 0.01, as determined by Student’s t-test.

FIGURE 8

Agronomic traits of transgenic lines overexpressing different OsGRF genes and working model for miR396-OsGRF in regulation of rice immunity and yield traits. Comparison of gross plant (A), scale bar = 16 cm, and panicle morphologies (B), scale bar = 5 cm, of EV (YB), OEGRF6, OEGRF8 and OEGRF9 transgenic plants. Comparison of gross plant (C), scale bar = 16 cm, and panicle morphologies (D), scale bar = 5 cm, of EV (YB), OEGRF7, and GRF7RNAi transgenic plants. Number of panicles (E), number of grains per panicle (F), and 1000-grain weight (G) of OsGRF6 over-expression lines. The asterisk above the bars indicate significant differences between EV and the indicated transgenic lines at a P-value < 0.01, as determined by Student’s t-test. (H) Working model for miR396-OsGRFs module in balancing yield traits and immunity against the blast fungus.