Rice Science ›› 2021, Vol. 28 ›› Issue (2): 156-165.DOI: 10.1016/j.rsci.2021.01.005
• Research Paper • Previous Articles Next Articles
Junhua Lu2, Xuemei Yang2, Jinfeng Chen2, Tingting Li2, Zijin Hu2, Ying Xie2, Jinlu Li2, Jiqun Zhao1,2, Mei Pu1,2, Hui Feng1,2, Jing Fan1,2, Yanyan Huang1,2, Jiwei Zhang1,2, Wenming Wang1,2, Yan Li1,2()
Received:
2019-10-03
Accepted:
2020-05-13
Online:
2021-03-28
Published:
2021-03-28
About author:
#These authors contributed equally to this work
Junhua Lu, Xuemei Yang, Jinfeng Chen, Tingting Li, Zijin Hu, Ying Xie, Jinlu Li, Jiqun Zhao, Mei Pu, Hui Feng, Jing Fan, Yanyan Huang, Jiwei Zhang, Wenming Wang, Yan Li. Osa-miR439 Negatively Regulates Rice Immunity Against Magnaporthe oryzae[J]. Rice Science, 2021, 28(2): 156-165.
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Fig. 1. Osa-miR439a is differentially responsive to Magnaporthe oryzae in susceptible and resistant rice accessions. A, Blast disease phenotype of representative leaf sections from Lijiangxintuanheigu (LTH) and International Rice Blast Line Pyricularia-Kanto51- m-Tsuyuake (IRBLkm-Ts) upon spray-inoculation of M. oryzae strain Guy11 (1 × 105 spore/mL) at 5 d post- inoculation (dpi). Scale bar, 1 cm. B, Accumulation of Osa-miR439a in LTH and IRBLkm-Ts upon M. oryzae/mock infection at indicated time points.C, Accumulation of Osa-miR439a in LTH with or without chitin treatment at indicated time points. hpi, Hours post-inoculation. In B and C, Error bars indicate SD (n = 3). Different letters above the bars indicate significant differences (P < 0.01) as determined by one-way analysis of variance followed by post hoc Tukey’s Honestly Significant Difference analysis.
Fig. 2. Overexpressing Osa-miR439a resulted in enhanced susceptibility to M. oryzae. A, Accumulation of Osa-miR439a in wild type (WT) control (Nipponbare) and the transgenic lines harboring 35S:miR439a (OX439a#1 and OX439a#2). B, Blast disease phenotypes on leaves of the indicated lines at 6 d post-inoculation of M. oryzae strain 97-27-2. Scale bar, 1 cm. C, Quantification analysis of the blast disease in B. Relative fungal biomass was determined by detecting the relative levels of M. oryzae Pot2 DNA against rice Ubiquitin DNA. D, Representative laser scanning confocal microscopy images showing the growth of the M. oryzae strain GZ8 at 24 and 36 h post- inoculation (hpi) on sheath cells of the indicated lines. AP, Appressorium; IH, Invasive hyphal. Scale bars, 20 µm. E, Quantification analysis of the progress of fungal infection at 24 and 36 hpi. Over 200 conidia in each line were analyzed. In A and C, error bars indicate SD (n = 3), and the letters above the bars indicate significant differences (P < 0.01) as determined by one- way analysis of variance followed by post hoc Tukey’s Honestly Significant Difference analysis.
Fig. 3. Overexpression of Osa-miR439a results in compromised disease-related defense responses. A and B, Expression of the defense-related genes (OsMAS1 and OsNAC4) in wild type (WT) and overexpression transgenic lines (OX439a) upon the infection of M. oryzae strain 97-27-2. RNA was extracted at the indicated time points for qRT-PCR assay. All the mRNA levels were normalized to that in the WT at 0 h post-inoculation (hpi). Error bars indicate SD (n = 3). Different lowercase letters above the bars indicate significant differences (P < 0.01) as determined by one-way analysis of variance followed by post hoc Tukey’s Honestly Significant Difference analysis.C, 3,3′-diaminobenzidine staining showing H2O2 accumulation at 2 d post-inoculation of M. oryzae strain 97-27-2. The photos at the top portion were taken with a stereo-microscope (Scale bars, 500 µm), while the photos at down portion were taken with a Zeiss-microscope (Zeiss imager A2) (Scale bars, 20 µm). AP, Appressorium.
Fig. 4. Overexpression of a target mimic of Osa- miR439a leads to compromised susceptibility to M. oryzae. A, Accumulation of Osa-miR439a in wild type (WT, Nipponbare) and transgenic lines expressing the target mimic of Osa-miR439a (MIM439). B, Blast disease phenotypes on leaves of the indicated lines at 7 d post-inoculation (dpi) of M. oryzae strain 97-27-2 (1 × 103 spore/mL). Scale bar, 1 cm. C, Quantification analysis of the blast disease in B. Relative fungal biomass was determined by detecting the expression level of M. oryzae Pot2 gene against rice Ubiquitin DNA level. D, 3,3′-diaminobenzidine staining showing H2O2 accumulation at 2 dpi of M. oryzae strain 97-27-2. The photos at the top portion were taken with a stereo-microscope (Scale bars, 500 µm). The photos at down portion were taken with a Zeiss- microscope (Zeiss imager A2) (Scale bars, 20 µm). AP, Appressorium. In A and C, error bars indicate SD (n = 3), and different lowercase letters above the bars indicate significant differences (P < 0.01) as determined by one-way analysis of variance followed by post hoc Tukey’s Honestly Significant Difference analysis.
Fig. 5. Expression of Osa-miR439a predicted target genes was differently responsive to M. oryzae in susceptible and resistant rice accessions. A, Graphs show the gene structure of the indicated Osa-miR439a target genes and the alignment of the target sequences with Osa-miR439a and MIM439 (target mimic of Osa-miR439a). Red lines indicate the target sites in the exon of target genes. Mismatched nucleotides are highlighted in red colors. UAU are insert bases.B and C, Relative mRNA levels of the target genes in the wild type (WT) and the indicated transgenic lines. D-F, Expression patterns of the indicated target genes in Lijiangxintuanheigu (LTH) and International Rice Blast Line Pyricularia-Kanto51-m- Tsuyuake (IRBLkm-Ts) upon M. oryzae infection. RNA was extracted at the indicated time points for qRT-PCR analysis. mRNA level was normalized to that in untreated samples (0 h post-inoculation, 0 hpi). In B-F, Different lowercase letters above the bars indicate significant differences (P < 0.01) as determined by one-way analysis of variance followed by post hoc Tukey’s Honestly Significant Difference (HSD) analysis. Error bars indicate SD (n = 3).
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