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.

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.

The spatiotemporal regulation of proteins and RNAs is essential for the precise development of reproductive tissues in many organisms. The anther, a significant part of the male reproductive organ in plants, contains the somatic cell layers named the anther wall and germ cells located inside. The cell-to-cell communication between the soma and germ cells in the development of male and female tissues is reported, although the mechanism remains unknown in plants. Here, we successfully developed a simple 3D-organ-immunoimaging technique, which distinguishes each single cell from the four somatic cell layers and germ cells without the need for transformation, embedding, sectioning, or clearing in rice. The 3D-immunostaining method is also applicable to the intracellular localization of meiosis-specific proteins in meiocytes-namely MEL1, a germ cell-specific Argonaute in the cytoplasm, and ZEP1, a pachytene marker on meiotic chromosomes. Our study suggests that the 3D-multiple immunostaining method with single-cell and intracellular resolution will contribute to the organ-level elucidation of comprehensive molecular mechanisms and non-cell-autonomous systems.