Identification and analysis of brown planthopper-responsive microRNAs in resistant and susceptible rice plants

Abstract

The brown planthopper (BPH) is the most devastating insect pest of rice. The rice gene BPH15 confers resistance to BPH. MicroRNAs (miRNAs) regulate a spectrum of development and defense response processes in plants. In this study, we analyzed six miRNA profiles of a BPH15 introgression line (P15) and a susceptible recipient line (PC) at three time points (0 h, 6 h and 48 h) after BPH attack, and identified 464 known miRNAs and 183 potential novel miRNAs. Before the BPH feeding, we identified 23 miRNAs differentially expressed in P15 and PC. We speculated that the resistant plant is in a priming state by the regulation of miRNAs. After the BPH feeding, 104 miRNAs were found to be expressed differentially in P15 (68 in P15-6/P15-0, 36 in P15-48/P15-0), and 80 miRNAs were found expressed differentially in PC (32 in PC-6/PC-0, 48 in PC-48/PC-0), which illustrated that miRNA expression is activated upon attack. These miRNAs regulate different pathways that contribute to the basal defense and specific resistance of rice to the BPH. Our study provides additional data for scientists to further explore the mechanism of plant defense against insect attack and to find a way for efficient insect control.

Figure 1

Size distribution and annotation of small RNAs from the libraries of the resistant BPH15 introgression line (P15) and susceptible recipient line (PC) at 0 h, 6 h, and 48 h after BPH infestation. (a) Length distribution of sequenced reads. The most abundant sRNAs in both libraries were 21 nt and 24 nt in length. (b) Proportions of different classes of small RNAs detected in the six libraries.

Figure 2

Contrast between up-regulated and down-regulated differentially expressed miRNAs in all comparisons. Differential expression analyses were run with the Bioconductor edgeR package with the condition that the ratio was greater than 2 and P < 0.05. Up represents the number of miRNAs that were up-regulated in the compared group, and down represents the number of miRNAs that were down-regulated in the compared group.

Figure 3

Stem-loop RT-PCR analysis of known miRNA levels in P15 and PC compared to sequencing results. The expression of miRNAs was normalized by small RNA U6. The data represent the mean ± SD from three biologically independent experiments. Statistical significance was analyzed using one-way ANOVA. The asterisks represent significance, where one asterisk indicates P ≤ 0.05, and two asterisks indicates P ≤ 0.01.

Figure 4

Stem-loop RT-PCR analysis of novel miRNA levels in P15 and PC compared to sequencing results. The expressions of miRNAs was normalized by small RNA U6. The data represent the mean ± SD from three biologically independent experiments. Statistical significance was analyzed using one-way ANOVA. The asterisks represent significance, where one asterisk indicates P ≤ 0.05, and two asterisks indicates P ≤ 0.01.

Figure 5

Regulatory network of two rice genotypes before BPH attack. ERF1: ethylene response factor 1; B-Glu11: beta glucosidase 11; oxidase: NADPH/respiratory burst oxidase protein D; PR5K: PR5-like receptor kinase; CSLD2: cellulose-synthase like D2; FER: malectin/receptor-like protein kinase family protein; Peroxidase: peroxidase superfamily protein; SHM1: serine transhydroxymethyltransferase 1; PIF3: phytochrome interacting factor 3; NB-ARC: NB-ARC domain-containing disease resistance protein; CTP synthase: CTP synthase family protein; isomerase: cyclophilin-like peptidyl-prolyl cis-trans isomerase family protein; oxidoreductase: FAD/NAD(P)-binding oxidoreductase.

Figure 6

Venn diagrams of differentially expressed miRNA candidates. Venn diagram of the number of differently expressed miRNA molecules of the resistant BPH15 introgression line (P15) and susceptible recipient line (PC) at 0 h, 6 h and 48 h after BPH infestation.

Figure 7

Venn diagram of common defense-related miRNAs in two rice genotypes 6 h and 48 h after BPH feeding.

Figure 8

Target assay of effect of miRNAs on the predicted target gene expression in rice protoplasts. (a) Fluorescence micrographs of rice protoplasts transfected with YFP, YFP/miR160f-5p, ARF16-YFP and ARF16-YFP/miR160f-5p from left to right. The photographs were taken at 60 × magnification. (b) Fluorescence micrographs of rice protoplasts transfected with YFP, YFP/miR167a-5p, NB-ARC-YFP and NB-ARC-YFP/miR167a-5p from left to right. The photographs were taken at 60 × magnification. (c) Western blot analysis of ARF16 expression or black plasmid expression in rice protoplasts transfected with YFP, YFP/miR160f-5p, ARF16-YFP and ARF16-YFP/miR160f-5p from left to right using anti-HA and anti-GAPDH antibodies. ARF16 is indicated with arrow. (d) Western blot analysis of NB-ARC expression or black plasmid expression in rice protoplasts transfected with YFP, YFP/miR167a-5p, NB-ARC-YFP and NB-ARC-YFP/miR167a-5p from left to right using anti-HA and anti-GAPDH antibodies. NB-ARC is indicated with arrow. The original blots of c and d are shown in Fig. S5.