Dicer-like 3 produces transposable element-associated 24-nt siRNAs that control agricultural traits in rice

Abstract

Transposable elements (TEs) and repetitive sequences make up over 35% of the rice (Oryza sativa) genome. The host regulates the activity of different TEs by different epigenetic mechanisms, including DNA methylation, histone H3K9 methylation, and histone H3K4 demethylation. TEs can also affect the expression of host genes. For example, miniature inverted repeat TEs (MITEs), dispersed high copy-number DNA TEs, can influence the expression of nearby genes. In plants, 24-nt small interfering RNAs (siRNAs) are mainly derived from repeats and TEs. However, the extent to which TEs, particularly MITEs associated with 24-nt siRNAs, affect gene expression remains elusive. Here, we show that the rice Dicer-like 3 homolog OsDCL3a is primarily responsible for 24-nt siRNA processing. Impairing OsDCL3a expression by RNA interference caused phenotypes affecting important agricultural traits; these phenotypes include dwarfism, larger flag leaf angle, and fewer secondary branches. We used small RNA deep sequencing to identify 535,054 24-nt siRNA clusters. Of these clusters, ∼82% were OsDCL3a-dependent and showed significant enrichment of MITEs. Reduction of OsDCL3a function reduced the 24-nt siRNAs predominantly from MITEs and elevated expression of nearby genes. OsDCL3a directly targets genes involved in gibberellin and brassinosteroid homeostasis; OsDCL3a deficiency may affect these genes, thus causing the phenotypes of dwarfism and enlarged flag leaf angle. Our work identifies OsDCL3a-dependent 24-nt siRNAs derived from MITEs as broadly functioning regulators for fine-tuning gene expression, which may reflect a conserved epigenetic mechanism in higher plants with genomes rich in dispersed repeats or TEs.

Keywords: plant architecture; plant hormone; transposon.

Fig. 1.

OsDCL3a knockdown plants display pleiotropic phenotypes affecting important agricultural traits. (A and B) OsDCL3a RNAi lines (3a-3 and 3a-1) show dwarf phenotypes (A) and statistical analysis of plant height (n = 30) (B). (C and D) Flag leaf inclination of WT, 3a-3, and 3a-1 (C) and statistical analysis of leaf angles (n = 30) (D). (E–G) Panicle morphology of WT, 3a-3, and 3a-1 (E) and statistical analysis of length (F) and branches of main panicle (G) (n = 30). **P < 0.01 with t test. Error bars correspond to the SD of biological repeats. (Scale bars: 10 cm in A and E; 2 cm in C.)

Fig. 2.

Distribution of OsDCL3a-dependent 24-nt siRNAs and different classes of repetitive sequences relative to up-regulated genes. (A) Pie chart showing the distribution of OsDCL3a-dependent 24-nt sRNAs loci based on Rice Genome Annotation Project Release 7 and miRBase release 20 annotations. (B) Differentially expressed genes in 3a-3 and 3a-1. Blue dots indicate up-regulated genes and green dots indicate down-regulated genes. (C) Abundance of 24-nt sRNAs in 2-kb regions upstream and downstream of up-regulated genes, with each gene annotated from the TSS to the TTS in WT (blue), 3a-3 (green), and 3a-1 (red). (D) The frequencies of four classes of repetitive sequences, MITEs (dark red), other DNA TEs (pink), retrotransposons (dark green), and other repeats (light green), were plotted in ±2 kb and gene body from TSS to TTS.

Fig. 3.

EUI encodes a GA deactivating enzyme and is activated in OsDCL3a RNAi lines. (A) Schematic representation of EUI. The arrowhead indicates the transcription start site. Boxes indicate exons (black), introns (white), UTR regions (gray), and MITEs (yellow). sRNA probes (SP5–SP7), bisulfite sequencing regions (BSP5, BSP6), and the regions used for ChIP-qPCR (R5–R7) are indicated by black lines. (B) sRNA-seq and RNA-seq data for EUI are shown in WT, 3a-3, and 3a-1. (C and D) Small RNA blot and qPCR validate the sRNA-seq and RNA-seq data, respectively. U6 probe and 5S rRNA/tRNA stained with ethidium bromide were used as small RNA blot loading controls. eEF1α was used as an internal reference for qPCR. The level of EUI transcription also was detected in ago4ab-1 and rdr2-2 lines. Error bars correspond to the SD. (E) Bisulfite sequencing analysis of the DNA methylation level of two MITE regions. (F) ChIP-qPCR assay detects the chromatin states using anti-H3K9me2. Anti-H3 was used as an internal reference for ChIP-qPCR. Error bars correspond to the SD.

Fig. 4.

24-nt siRNAs from MITEs and tandem repeats regulate expression of a nearby gene, Os08g19420. (A) Schematic of Os08g19420 with putative MITE Ditto-like and tandem repeats indicated by yellow boxes and arrows within the white box, respectively. Green boxes indicate LTR, En/Spm-like TEs, and a SINE element from left to right. Boxes indicate exons (black), introns (white), and UTR regions (gray). Black lines show the positions of siRNA probes (SP10–SP14), bisulfite sequencing regions (BSP9, BSP10), and five ChIP-qPCR analysis regions (R11–R17). (B) Small RNA-seq and RNA-seq data are shown in the region of Os08g19420 from WT (blue), 3a-3 (green), and 3a-1 (red). (C) Detection of 24-nt siRNAs by small RNA blot in WT, 3a-3, and 3a-1. (D) qPCR validation of Os08g19420 expression in WT, 3a-3, 3a-1, ago4ab-1, and rdr2-2 plants were normalized using the signal from eEF1α gene. The average ± SD values from three biological repeats are shown. (E) Bisulfite sequencing analysis of the DNA methylation level of MITE and tandem repeats. (F) In WT, 3a-3, and 3a-1, the chromatin states are detected by anti-H3K9me2 ChIP-qPCR assays at seven different regions. Anti-H3 was used as internal reference. Error bars correspond to the SD.

Fig. 5.

OsDCL3a-dependent 24-nt siRNAs regulate nearby gene expression and control rice development. In WT plants (A), 24-nt siRNAs (red lines) produced from TEs and repetitive sequences (yellow triangles) target nearby genes, including genes involved in GA and BR homeostasis. Normal regulation of GA and BR produces normal morphology. OsDCL3a RNAi knockdown (KD) plants (B) produce fewer 24-nt siRNAs (red lines), causing increased expression of genes involved in GA and BR homeostasis. Perturbed regulation of GA and BR biosynthetic genes results in an imbalance of GA and BR and causes abnormal morphology.