Integrated Analysis of MicroRNA and Target Genes in Brachypodium distachyon Infected by Magnaporthe oryzae by Small RNA and Degradome Sequencing

Abstract

Rice blast caused by Magnaporthe oryzae is one of the most important diseases that seriously threaten rice production. Brachypodium distachyon is a grass species closely related to grain crops, such as rice, barley, and wheat, and has become a new model plant of Gramineae. In this study, 15 small RNA samples were sequenced to examine the dynamic changes in microRNA (miRNA) expression in B. distachyon infected by M. oryzae at 0, 24, and 48 h after inoculation. We identified 432 conserved miRNAs and 288 predicted candidate miRNAs in B. distachyon. Additionally, there were 7 and 19 differentially expressed miRNAs at 24 and 48 h post-inoculation, respectively. Furthermore, using degradome sequencing, we identified 2,126 genes as targets for 308 miRNAs; using quantitative real-time PCR (qRT-PCR), we validated five miRNA/target regulatory units involved in B. distachyon-M. oryzae interactions. Moreover, using co-transformation technology, we demonstrated that BdNAC21 was negatively regulated by miR164c. This study provides a new approach for identifying resistance genes in B. distachyon by mining the miRNA regulatory network of host-pathogen interactions.

Keywords: Brachypodium distachyon; Magnaporthe oryzae; high-throughput sequencing; miRNA; target.

FIGURE 1

Profiling of small RNA sequencing in B. distachyon plants infected by M. oryzae. (A) Total clean reads-distribution and radioactivity (% of total) in libraries at each time point between each experimental group and the control group. (B) The classification distribution of the total small RNA in the five B. distachyon small RNA libraries.

FIGURE 2

Total abundance of small RNA sequences in each size class and miRNA nucleotide bias analysis results. (A) Length distribution of sequencing results (total). (B) Results of total miRNA nucleotide bias. bdi is short for Brachypodium distachyon; gma is short for Glycine max; mdm is short for Malus domestica; osa is short for Oryza sativa; ptc is short for Populus trichocarpa; ath is short for Arabidopsis thaliana; mtr is short for Medicago truncatula; ata is short for Aegilops tauschii; gra is short for Gossypium raimondii; hvu is short for Hordeum vulgare; lja is short for Lotus japonicus; far is short for Festuca arundinacea. (C) Results of miRNA first nucleotide bias at each position.

FIGURE 3

The statistics of miRNAs in the disparate sample. (A) Number of identified known and novel miRNAs. (B) Distribution of miRNA counts in different plant species. (C) Summary of the number of miRNA family members.

FIGURE 4

Venn-diagram analysis of the number and overlap of miRNAs in B. distachyon plants infected by M. oryzae at 0, 24, and 48 hpi.

FIGURE 5

Heatmaps of differentially expressed miRNAs. (A) Heatmaps of differentially expressed miRNAs of B. distachyon leaves infected by M. oryzae at 24 hpi. (B) Heatmaps of differentially expressed miRNAs of B. distachyon leaves infected by M. oryzae at 48 hpi.

FIGURE 6

Gene Ontology (GO) term enrichments at 24 (A) and 48 (B) hpi.

FIGURE 7

(A–E) qRT-PCR analysis of the expression of miRNAs and their corresponding targets during B. distachyon–M. oryzae interaction. The expression of mRNAs and their corresponding targets were analyzed by qRT-PCR. Asterisk (*) represents statistically significant difference (P < 0.05). Blue columns indicate miRNAs, and red columns indicate their corresponding targets. Expression values were normalized by UBC18 expression.

FIGURE 8

Co-transformation of BdNAC21 and miR164c in N. benthamiana leaves. Recombinant vectors were carried into N. benthamiana leaves by Agrobacterium strain GV3101. (A) Histochemical staining of GUS activity. (B) Quantitative analysis of GUS fluorescence.