MicroRNA-mediated regulation of gene expression in the response of rice plants to fungal elicitors

Abstract

MicroRNAs (miRNAs) are small non-coding RNAs that have important regulatory functions in plant growth, development, and response to abiotic stress. Increasing evidence also supports that plant miRNAs contribute to immune responses to pathogens. Here, we used deep sequencing of small RNA libraries for global identification of rice miRNAs that are regulated by fungal elicitors. We also describe 9 previously uncharacterized miRNAs in rice. Combined small RNA and degradome analyses revealed regulatory networks enriched in elicitor-regulated miRNAs supported by the identification of their corresponding target genes. Specifically, we identified an important number of miRNA/target gene pairs involved in small RNA pathways, including miRNA, heterochromatic and trans-acting siRNA pathways. We present evidence for miRNA/target gene pairs implicated in hormone signaling and cross-talk among hormone pathways having great potential in regulating rice immunity. Furthermore, we describe miRNA-mediated regulation of Conserved-Peptide upstream Open Reading Frame (CPuORF)-containing genes in rice, which suggests the existence of a novel regulatory network that integrates miRNA and CPuORF functions in plants. The knowledge gained in this study will help in understanding the underlying regulatory mechanisms of miRNAs in rice immunity and develop appropriate strategies for rice protection.

Keywords: Magnaporthe oryzae; conserved peptide upstream open reading frame; degradome; elicitors; miRNAs; rice.

Figure 1.

Expression profiling of known miRNAs from rice. (A) Expression of known miRNAs in leaves and roots of rice plants. Reads retrieved from the Solexa/Illumina sequencing dataset for each family member in control libraries were normalized to the total count of reads obtained in the corresponding library. Only the most abundantly expressed miRNAs are presented. Asterisks denote miRNAs examined in (B and C). Details on miRNA expression in each tissue are in Supplemental Table 1. (B, C) Expression of miRNAs identified in small RNA libraries from rice tissues by Northern blot analysis (B) or stem-loop RT-qPCR (C). Lower panel in (B) shows ethidium bromide staining of RNA samples. Oligonucleotides used as probes in (B) are indicated on the right side.

Figure 2.

Precursor structures of novel members of known miRNA families. Small RNA sequences recovered from the Solexa/Illumina sequencing data mapping into these structures are represented by black bars. The nucleotide sequences of these precursor structures are in Supplementary Figure 2.

Figure 3.

Precursor structures and detection of novel miRNAs from rice. (A) Precursor structures of novel miRNAs. Small RNA sequences mapping into these structures are represented by black bars. Additional information on the nucleotide sequence and chromosomal location is in Supplemental Figure 3. (B) Northern blot analysis of novel miRNAs. Total RNA samples (200 µg) were analyzed. The same blots were successively stripped and re-probed withP-end-labeled oligonucleotides. Except for miR11341, which was detected in roots, all novel miRNAs accumulated in rice leaves. Lower panels show ethidium bromide staining of RNA samples.

Figure 4.

Elicitor-responsiveness of known miRNAs from rice. (A) Expression analysis of known miRNAs in leaves and roots at 30 min or 2 h of elicitor treatment (light and dark bars, respectively) as determined by the logarithm of fold change (elicitor vs. control). Representative examples are shown (see Table S1 for detailed information on the expression of the complete list of rice miRNAs). Asterisks denote miRNAs examined in (B). (B) Northern blot analysis of miR156a, miR529b and miR5078 in control and elicitor-treated rice leaves. Total RNA samples (70 µg) were analyzed. Oligonucleotides used as probes are indicated on the right. c, control; e, elicitor.

Figure 5.

Confirmed target genes for rice miRNA using degradome sequencing. (A) Pipeline for target identification in degradome sequencing. (B) MapMan annotation of miRNA targets validated by degradome analysis in control and elicitor-treated leaves. Only the validated target genes identified by degradome sequencing in categories 0, 1 and 2 were considered. CM, carbohydrate metabolism. (C) Schematic of the Conserved-Peptide upstream Open Reading Frame (CPuORF3)-containing OsbZIP38 gene targeted by miR5819. Validation by 5′RACE of miRNA-mediated cleavage of CPuORF3-containing bZIP transcripts is shown.

Figure 6.

Overview of regulatory networks in which miRNA/target pairs function during the rice response to fungal elicitors. Small RNA and degradome sequencing data were used to establish regulations in the indicated pathways. All target genes were supported by degradome sequencing. miRNAs targeting these genes are boxed. Except for EDF1, all the indicated target genes are classified in categories 0, 1 or 2 in the degradome analysis. (A) Small RNA biogenesis and functioning. AGO, ARGONAUTE; DCL, DICER-like; RDR, RNA-dependent RNA polymerase; SGS3, Suppressor of gene silencing. (B) Ethylene signaling pathway and crosstalk with salcylic-acid and jasmonic-acid signaling pathways, and polyamine biosynthesis. EDF1, ethylene response DNA binding factor 1; EREBP, ethylene-responsive element binding protein; ERF, ethylene responsive factor; ETR, ethylene receptor; JMT, jasmonic acid carboxyl methyltransferase; Me-JA, methyl-jasmonic acid; Me-SA, methyl-salicylic acid; SAM, S-adenosylmethionine; SAMT, salicylic acid carboxyl methyltransferase. (C) Auxin signaling pathway. ACC, 1-aminocyclopropane-1-carboxylate; ACO, ACC oxidase; ARF, auxin response factor; TIR1, TRANSPORT INHIBITOR RESPONSE 1.

Figure 7.

miR5819-mediated regulation of OsbZIP38 in rice in response to treatment with fungal elicitors. (A) Alignment of the conserved Sucrose Control-uORF (SC-uORF) amino acid sequences present in the 5′ UT region of the group S bZIP-type transcription factors in Arabidopsis (AtbZIP11 and AtbZIP2), and rice miRNA-regulated bZIP transcription factors identified in this study (OsbZIP38, OsbZIP27). Dark and light gray indicate different amino acids. (B) A model depicting the regulation of OsbZIP38 expression by miR5819 and CPuORFs. Treatment with fungal elicitors regulates miR5819 accumulation, which in turn negatively regulates the accumulation of OsbZIP38 transcripts. The target site of miR5819 locates at the nucleotide sequence encoding the short peptide (sPEP, encoded by CPuORF3). Sucrose can modulate translation of AtbZIP11 and AtbZIP2 (members of the S1-group of bZIP transcription factors) via SC-uORF (Wiese et al. 2004). Whether OsbZIP38 is translationally controlled by sucrose remains to be determined.