Spatial distribution of three ARGONAUTEs regulates the anther phasiRNA pathway.

Argonaute protein (AGO) in association with small RNAs is the core machinery of RNA silencing, an essential mechanism for precise development and defense against pathogens in many organisms. Here, we identified two AGOs in rice anthers, AGO1b and AGO1d, that interact with phased small interfering RNAs (phasiRNAs) derived from numerous long non-coding RNAs. Moreover, 3D-immunoimaging and mutant analysis indicated that rice AGO1b and AGO1d cell type-specifically regulate anther development by acting as mobile carriers of these phasiRNAs from the somatic cell layers to the germ cells in anthers.

3D multiple immunoimaging using whole male organs in rice.

Spatiotemporal regulation of proteins and RNAs is essential for the precise development of reproductive tissues in many organisms. The anther, a prominent part of the male reproductive organ in plants, contains several somatic cell layers named the anther wall and, within it, the germ cells. Here, we successfully developed a simple 3D organ-immunoimaging technique for rice anthers, which distinguishes each individual cell from the four somatic cell layers and germ cells without the need for transformation, embedding, sectioning, or clearing.

3D Imaging and In Situ Hybridization for Uncovering the Functions of MicroRNA in Rice Anther.

Small RNAs specifically expressed in reproductive tissues are key regulators of germline development in eukaryotes. Rice microRNA2118 (miR2118), which is enriched during reproduction in grasses, is a trigger to produce phased small interfering RNAs (phasiRNAs). These phasiRNAs demonstrate the temporal regulation with premeiotic phasiRNAs and meiotic phasiRNAs in anther development.

Spatiotemporal regulation and roles of reproductive phasiRNAs in plants.

Since co-suppression was discovered as a pioneer silencing phenomenon of RNA interference (RNAi) in petunia in 1990, many types of small RNAs have been identified in the RNAi pathway among various eukaryotes. In plants, a large number of 21- or 24-nucleotide (nt) phased small interfering RNAs (phasiRNAs) are produced via processing of long RNA precursors by Dicer-like proteins. However, the roles of phasiRNAs remain largely unknown.

miR2118-dependent U-rich phasiRNA production in rice anther wall development.

Reproduction-specific small RNAs are vital regulators of germline development in animals and plants. MicroRNA2118 (miR2118) is conserved in plants and induces the production of phased small interfering RNAs (phasiRNAs). To reveal the biological functions of miR2118, we describe here rice mutants with large deletions of the miR2118 cluster.

Biogenesis of diverse plant phasiRNAs involves an miRNA-trigger and Dicer-processing.

It has been almost 30 years since RNA interference (RNAi) was shown to silence genes via double-stranded RNAs (dsRNAs) in Caenorhabditis elegans (Fire et al. 1998). 20-30-nucleotide (nt) small non-coding RNAs are a key element of the RNAi machinery.

Rice germline-specific Argonaute MEL1 protein binds to phasiRNAs generated from more than 700 lincRNAs.

Small RNAs that interact with Argonaute (AGO) proteins play central roles in RNA-mediated silencing. MEIOSIS ARRESTED AT LEPTOTENE1 (MEL1), a rice AGO, has specific functions in the development of pre-meiotic germ cells and the progression of meiosis. Here, we show that MEL1, which is located mostly in the cytoplasm of germ cells, associates preferentially with 21-nucleotide phased small interfering RNAs (phasiRNAs) that bear a 5'-terminal cytosine.

Isolation and bioinformatic analyses of small RNAs interacting with germ cell-specific argonaute in rice.

The small noncoding RNAs in plants are categorized into two major classes, 21-nucleotides (nt) micro RNA (miRNA) and 21- or 24-nt small-interfering RNA (siRNA). ARGONAUTE (AGO) proteins associate with small RNAs and play central roles in transcriptional and posttranscriptional gene regulation. In plants, AGO1-miRNA complexes mainly regulate developmental processes, and AGO4-siRNA complexes suppress the activity of transposons and exogenous viral infections via RNA-directed DNA methylation.

A gene network for long-day flowering activates RFT1 encoding a mobile flowering signal in rice.

Although some genes that encode sensory or regulatory elements for photoperiodic flowering are conserved between the long-day (LD) plant Arabidopsis thaliana and the short-day (SD) plant rice, the gene networks that control rice flowering, and particularly flowering under LD conditions, are not well understood. We show here that RICE FLOWERING LOCUS T 1 (RFT1), the closest homolog to Heading date 3a (Hd3a), is a major floral activator under LD conditions. An RFT1:GFP fusion protein localized in the shoot apical meristem (SAM) under LD conditions, suggesting that RFT1 is a florigen gene in rice.

Hd3a and RFT1 are essential for flowering in rice.

RICE FLOWERING LOCUS T 1 (RFT1/FT-L3) is the closest homologue of Heading date 3a (Hd3a), which is thought to encode a mobile flowering signal and promote floral transition under short-day (SD) conditions. RFT1 is located only 11.5 kb from Hd3a on chromosome 6.