Tamotsu, H. | bioRxiv Science

bioRxiv2022

Spatial control of ARGONAUTE-mediated RNA silencing in anther development

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 demonstrated that rice AGO1b and AGO1d redundantly and 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.

plant biology↗

bioRxiv2022

3D-multiple immunoimaging using whole male organs in rice

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.

plant biology↗