21-nt phasiRNAs direct target mRNA cleavage in rice male germ cells

In grasses, phased small interfering RNAs (phasiRNAs), 21- or 24-nucleotide (nt) in length, are predominantly expressed in anthers and regulate male fertility. However, their targets and mode of action on the targets remain unknown. Here we profile phasiRNA expression in premeiotic and meiotic spikelets as well as in purified male meiocytes at early prophase I, tetrads and microspores in rice. We show that 21-nt phasiRNAs are most abundant in meiocytes at early prophase I while 24-nt phasiRNAs are more abundant in tetrads and microspores. By performing highly sensitive degradome sequencing, we find that 21-nt phasiRNAs direct target mRNA cleavage in male germ cells, especially in meiocytes at early prophase I. The target genes in early prophase I meiocytes show an enrichment for carbohydrate biosynthetic and metabolic pathways. Our study provides strong evidence that 21-nt phasiRNAs act in a target-cleavage mode and may facilitate the progression of meiosis by fine-tuning carbohydrate biosynthesis and metabolism in male germ cells. Overall design: To profile phasiRNAs and explore the potential targets of phasiRNAs in male germ cells at different stages, we collected highly pure meiocytes at early prophase I tetrads, and microspores, respectively, by micromanipulation. Pre-meiotic spikelets, in which anthers are at stage 2 to stage 3 , meiotic spikelets in which germ cells are mostly at the early prophase of meiosis I, and embryos from seeds germinated 24h at 28?, were also collected as controls. We then using these samples performed low-input sRNA-seq, sRNA-seq and high sensitive degradome-seq in three biological replicates.