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. Our study also highlights a new mode of reproductive RNA silencing via the specific nuclear and cytoplasmic localization of three AGOs, AGO1b, AGO1d, and MEL1, in rice pollen mother cells.

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. The 3D immunostaining method is also applicable to the intracellular localization of meiosis-specific proteins in meiocytes, as exemplified by MEL1, a germ cell-specific ARGONAUTE in the cytoplasm, and ZEP1, a pachytene marker on meiotic chromosomes. Our 3D multiple immunostaining method with single-cell and intracellular resolution will contribute to a comprehensive organ-level elucidation of molecular mechanisms and cellular connectivity.

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. Furthermore, the site-specific regulation via miR2118 and phasiRNAs is of importance in soma and germ development in anthers. Accordingly, histological imaging methods are essential tools for understanding spatiotemporal regulation during reproduction and elucidating the reproductive roles of miRNAs and phasiRNAs. We successfully developed a method to visualize the three-dimensional (3D) structure of entire rice anthers, which can also be used for distinguishing the internal structure of the anthers in other plants. Here, we describe the detailed methods of in situ hybridization for miR2118 localization and the visualization of the 3D structure of entire anthers of rice.

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. The development of imaging technology and RNA profiling has clarified the spatiotemporal regulation of phasiRNAs, and subsequently the different functions of 21-nt trans-acting phasiRNAs and 24-nt cis-regulatory phasiRNAs during male organ development. This review focuses on the biogenesis, diversification, spatiotemporal expression pattern and function of phasiRNAs in plants.

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. Our results demonstrate that the loss of miR2118 causes severe male and female sterility in rice, associated with marked morphological and developmental abnormalities in somatic anther wall cells. Small RNA profiling reveals that miR2118-dependent 21-nucleotide (nt) phasiRNAs in the anther wall are U-rich, distinct from the phasiRNAs in germ cells. Furthermore, the miR2118-dependent biogenesis of 21-nt phasiRNAs may involve the Argonaute proteins OsAGO1b/OsAGO1d, which are abundant in anther wall cell layers. Our study highlights the site-specific differences of phasiRNAs between somatic anther wall and germ cells, and demonstrates the significance of miR2118/U-phasiRNA functions in anther wall development and rice reproduction.

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. Recently, phased small interfering RNAs (phasiRNAs), small RNAs that are generated from a long RNA precursor at intervals of 21 to 26-nt, have been identified in plants and animals. In Drosophila, phasiRNAs are generated by the endonuclease, Zucchini (Zuc), in germlines. These phasiRNAs, known as one of PIWI-interacting RNAs (piRNAs), mainly repress transposable elements. Similarly, reproduction-specific phasiRNAs have been identified in the family Poaceae, although DICER LIKE (DCL) protein-dependent phasiRNA biogenesis in rice is distinct from piRNA biogenesis in animals. In plants, phasiRNA biogenesis is initiated when 22-nt microRNAs (miRNAs) cleave single-stranded target RNAs. Subsequently, RNA-dependent RNA polymerase (RDR) forms dsRNAs from the cleaved RNAs, and dsRNAs are further processed by DCLs into 21 to 24-nt phasiRNAs. Finally, the phasiRNAs are loaded to ARGONAUTE (AGO) proteins to induce RNA-silencing. There are diverse types of phasiRNA precursors and the miRNAs that trigger the biogenesis. Their expression patterns also differ among plant species, suggesting that species-specific combinations of these triggers dictate the spatio-temporal pattern of phasiRNA biogenesis during development, or in response to environmental stimuli.

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. Most phasiRNAs are derived from 1171 intergenic clusters distributed on all rice chromosomes. From these clusters, over 700 large intergenic, non-coding RNAs (lincRNAs) that contain the consensus sequence complementary to miR2118 are transcribed specifically in inflorescences, and cleaved within the miR2118 site. Cleaved lincRNAs are processed via DICER-LIKE4 (DCL4) protein, resulting in production of phasiRNAs. This study provides the evidence that the miR2118-dependent and the DCL4-dependent pathways are both required for biogenesis of 21-nt phasiRNAs associated with germline-specific MEL1 AGO in rice, and over 700 lincRNAs are key factors for induction of this biogenesis during reproductive-specific stages.

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. In many animal species, the PIWI-subfamily AGOs interact with PIWI-interacting RNAs (piRNAs), which are most commonly 24-34 nt, and function to tame transposons and to regulate mRNA translation and stability in the germline. The rice protein MEIOSIS ARRESTED AT LEPTOTENE1 (MEL1) is a plant AGO member that has roles specific to development and maintenance of germ cells before meiosis. MEL1-binding small RNAs are mainly 21 nt, have a 5'-terminal cytosine, and are distinct from animal piRNAs. In this chapter, we describe methods for RNA-immunoprecipitation (RNA-IP) using a specific antibody that recognizes MEL1 and subsequent purification of MEL1-associating small RNAs from the IP fraction. We also introduce the bioinformatic procedures including mapping, annotation, and identifying small RNA clusters on the rice genome.

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. Furthermore, mutants in OsMADS50, a rice ortholog of Arabidopsis SUPPRESOR OF OVEREXPRESSION OF CONSTANS 1 (SOC1) did not flower up to 300 days after sowing under LD conditions, indicating that OsMADS50, which acts upstream of RFT1, promotes flowering under LD conditions. We propose that both positive (OsMADS50 and Ehd1) and negative (Hd1, phyB and Ghd7) regulators of RFT1 form a gene network that regulates LD flowering in rice. Among these regulators, Ehd1, a rice-specific floral inducer, integrates multiple pathways to regulate RFT1, leading to flowering under appropriate photoperiod conditions. A rice ortholog of Arabidopsis APETALA1, OsMADS14, was expressed in the floral meristem in wild-type but not in RFT1 RNAi plants, suggesting that OsMADS14 is activated by RFT1 protein in the SAM after the transition to flowering. We have thus exposed a network of genes that regulate LD flowering 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. Although RFT1 RNAi plants flowered normally, double RFT1-Hd3a RNAi plants did not flower up to 300 days after sowing (DAS), indicating that Hd3a and RFT1 are essential for flowering in rice. RFT1 expression was very low in wild-type plants, but there was a marked increase in RFT1 expression by 70 DAS in Hd3a RNAi plants, which flowered 90 DAS. H3K9 acetylation around the transcription initiation site of the RFT1 locus had increased by 70 DAS but not at 35 DAS. In the absence of Hd3a and RFT1 expression, transcription of OsMADS14 and OsMADS15, two rice orthologues of Arabidopsis APETALA1, was strongly reduced, suggesting that they act downstream of Hd3a and RFT1. These results indicate that Hd3a and RFT1 act as floral activators under SD conditions, and that RFT1 expression is partly regulated by chromatin modification.