GSE191268
Temperature-sensitive sterility in rice determined by the dual role of AGO1d in phasiRNA biogenesis and function
Phased secondary siRNAs (phasiRNAs) are broadly present in the reproductive tissues of flowering plants. Monocots generate 21- and 24-nt phasiRNAs by miR2118 and miR2275 respectively from noncoding RNAs with spatial-temporal specificity during anther development. However, which ARGONAUTES (AGOs) associate with these two miRNAs remains elusive. Here we show that rice AGO1d, specifically expressed in anther wall cells before and during meiosis, associates with both miR2118 and miR2275 to mediate phasiRNA biogenesis. Furthermore, AGO1d also binds to miR2118-triggered 21-nt phasiRNAs preferentially with a 5'-terminal uridine, suggesting a dual role in phasiRNA biogenesis and function. Depletion of AGO1d causes reduction of 21- and 24-nt phasiRNAs and temperature-sensitive sterility. Anthers of the ago1d mutants at lower temperatures mainly display defects in tapetal cells and vacuolation during pollen formation. These results uncover an essential role of AGO1d in rice anther development at lower temperatures and demonstrate coordinative roles of AGO proteins during reproductive phasiRNA biogenesis and function Overall design: For RNA-seq, rice anthers at stages of 4-6, 7-8, and 9 from both wild-type and ago1d mutant grown at 28 °C and 22°C were collected with three biological replicates. Total RNA was isolated from each sample and then used for poly(A) RNA isolation with oligo(dT) beads. Poly(A) RNA samples were then used for RNA-seq library construction using NEBNext UltraTM RNA Library Prep Kit for Illumina (New England Biolabs, USA) following manufacturer's recommendations. RNA-seq libraries were sequenced on an Illumina NovaSeq 6000 platform at Novogene. For small RNA-seq, anthers at stages of 4-6, 7-8, and 9 from both wild-type and ago1d mutant grown at 28 °C and 22°C were collected with three biological replicates. The small RNA libraries were prepared from a total of 2 µg total RNA isolated from each sample using NEBNext Multiplex Small RNA Library Prep Set for Illumina (New England Biolabs, USA) following manufacturer's recommendations and index codes were added to attribute sequences to each sample. All the small RNA libraries were sequenced on an Illumina novaseq 6000 platform at Novogene.For the AGO1d-FLAG RIP-seq, three biological replicates of rice spikelets at developmental stages of 4-6, 7-8, and 9 from AGO1d::AGO1d-FLAG transgenic plants were collected,  and grounded in liquid nitrogen into fine powder. The grounded powder was homogenized in 4 uL extraction buffer [50 mM Tris–HCl pH 7.5, 150 mM NaCl, 0.1% NP-40, 4 mM MgCl2, 5 mM DTT, EDTA-free protease inhibitor cocktail (Roche), and 400 U RNasin Ribonuclease Inhibitor (Promega)] and centrifuged at 12,000 g for 10 min at 4°C. 200 µL of the supernatant was aliquoted for RNA extraction using TRIzol reagent and used as input for RIP-seq. The rest of supernatant was precleared with Protein A/G magnetic beads (MedChemExpress, USA) at 4 °C for 1 h and then incubated with the anti-FLAG antibody (Cat. M185-3L, MBL, Japan) at 4°C for 2 h. Thereafter, 50 µL Protein A/G magnetic beads were added in and incubated at 4°C for 1 h. After 3 times of wash with the extraction buffer containing 2 mM DTT at 4°C, magnetic beads were extracted using TRIzol reagent for RNA isolation. Precipitated RNA was then used for small RNA library construction using the NEBNext Multiplex Small RNA Library Prep Set for Illumina (New England Biolabs, USA). For the RIP-seq input library construction, total RNA was loaded on a 15% urea PAGE gel for electrophoresis with the small-RNA Ladders (New England Biolabs, USA). Small RNA in ~15- to 30-nt was extracted from gel slices and used for input library construction. All the small RNA libraries were sequenced on an Illumina novaseq 6000 platform at Novogene, China.