Biology subjects

Nagahara, S.

Publications and source records attributed to Nagahara, S..

5 recordsLinked to original sources

bioRxiv2026

EC1 body controls sperm cell reception via gamete pit formation.

The remarkable efficiency of double fertilization in flowering plants depends on the precise delivery of immotile sperm cells to two female gametes1. Although sperm cells are rapidly released from the pollen tube, how they are accurately directed to their fertilization site has remained unknown. Here we identify a transient extracellular structure, termed the gamete pit, that forms at the egg cell-central cell (EC-CC) interface immediately after pollen tube discharge and serves as the landing site for sperm cells. Gamete pit formation is enabled by localized dissociation of the EC-CC interface, mediated by extracellular protein assemblies designated EC1 bodies. These structures contain extracellular deposits enriched in EGG CELL 1 (EC1) peptides2, including amyloid-like assemblies. Loss of EC1 bodies prevented gamete pit formation, causing sperm-cell backflow or catastrophic over-penetration of pollen tube contents into the central cell. Surprisingly, ectopic deposition of a heterologous amyloidogenic protein alleviated over-penetration and partially restored fertility. Our findings reveal a mechanism by which extracellular protein assemblies remodel the gamete interface for sperm-cell reception, uncover an unexpected structural role for the conserved gamete-activating peptide EC1, and establish protein-mediated cell-interface remodeling as a fundamental principle underlying double fertilization in flowering plants.

plant biology↗

bioRxiv2024

Development of a Versatile System to Facilitate Targeted Knockout/Elimination Using CRISPR/Cas9 for Highly Duplicated Gene Families in Arabidopsis Sexual Reproduction

CRISPR/Cas9-based targeted gene editing is a fundamental technique for studying gene functions in various organisms. In plants, the introduction of a T-DNA construct harboring Cas9 nuclease and single guide RNA (sgRNA) sequences induces sequence-specific DNA double-strand breaks, inducing the loss of gene function. Arabidopsis thaliana is a model for CRISPR/Cas9 system development and gene function studies; the introduction of Cas9 under the egg or zygote promoter and multiple sgRNA modules generates heritable or non-mosaic mutants for multiple targets in the T1 generation of A. thaliana. Recent reports reflect use of several CRISPR/Cas9 vectors in generating single- and higher-order mutants; however, the development of a reliable, cost-effective, and high-throughput CRISPR/Cas9 platform is necessary for targeting highly duplicated gene families. In this study, we have developed a simple and user-friendly construction system for the CRISPR/Cas9 vector series with improved gene editing efficiency by simply inserting a single intron into Cas9, and effectively demonstrated the simultaneous knockout of multiple genes involved in A. thaliana sexual reproduction. An unbiased PCR-mediated mutant identification in the T1 generation revealed that our CRISPR/Cas9 system can support a > 70 kb deletion of > 30 tandemly duplicated synergid-specific genes and simultaneous knockout of five redundant genes essential for double fertilization. We performed a one-shot knockout of seven homologous pollen tube receptor-like kinase genes and identified their specific and overlapping roles in pollen tube growth and guidance. Our system can potentially facilitate further research in experimental plant biology to search for genetically unidentified components using reverse genetic candidate approaches.

plant biology↗

bioRxiv2023

The Chaperone NASP Contributes to De Novo Deposition of the Centromeric Histone Variant CENH3 in Arabidopsis Early Embryogenesis

The centromere is an essential chromosome region where the kinetochore is formed to control equal chromosome distribution during cell division. The centromere-specific histone H3 variant CENH3 (also called CENP-A) is a prerequisite for the kinetochore formation. Since CENH3 evolves rapidly, associated factors, including histone chaperones mediating the deposition of CENH3 on the centromere, are thought to act through species-specific amino-acid sequences. The functions and interaction networks of CENH3 and histone chaperons have been well-characterized in animals and yeasts. However, molecular mechanisms involved in recognition and deposition of CENH3 are still unclear in plants. Here, we used a swapping strategy between domains of CENH3 of Arabidopsis thaliana and the liverwort Marchantia polymorpha to identify specific regions of CENH3 involved in targeting the centromeres and interacting with the general histone H3 chaperone, NASP (nuclear autoantigenic sperm protein). CENH3s LoopN-1 region was necessary and sufficient for the centromere targeting in cooperation with the 2 region and was involved in interaction with NASP in cooperation with N, suggesting a species-specific CENH3 recognition. In addition, by generating an Arabidopsis nasp knockout mutant in the background of a fully fertile GFP-CENH3/cenh3-1 line, we found that NASP was implicated for de novo CENH3 deposition after fertilization and thus for early embryo development. Our results imply that the NASP mediates the supply of CENH3 in the context of the rapidly evolving centromere identity in land plants.

plant biology↗

bioRxiv2023

Deep imaging revealed dynamics and signaling in one-to-one pollen tube guidance

In angiosperms, pollen tube guidance allows sperm cell delivery to the female gametes within the ovule, which are deeply embedded in a flower. However, when an ovary includes multiple pollen tubes and ovules, it is unclear how each ovule is fertilized one-to-one by a pollen tube. Here, our two-photon imaging revealed the pollen tube dynamics in living ovaries. The number of pollen tubes and ovule maturity affected the target selection among multiple ovules. On the inner surface of the septum epidermis within the transmitting tract, pollen tube behavior and emergence were regulated by the ovular sporophytic signals. In funicular guidance, the second pollen tube was strictly repelled by the FERONIA and LORELEI-dependent gametophytic signal, especially more than 45 minutes after the first pollen tube had passed. Such highly spatiotemporal regulation mechanisms in the one-to-one pollen tube guidance may allow angiosperms to produce more offspring in nature.

plant biology↗

bioRxiv2021

Detection of a biolistic delivery of fluorescent markers and CRISPR/Cas9 to the pollen tube

In recent years, genome-editing techniques, such as the CRISPR/Cas9 system, have been highlighted as a new approach to plant breeding. Agrobacterium-mediated transformation has been widely utilized to generate transgenic plants by introducing plasmid DNA containing CRISPR/Cas9 into plant cells. However, this method is generally applicable to a limited range of plants, such as model species. To overcome this limitation, we developed a method to genetically modify male germ cells without the need for Agrobacterium-mediated transfection and tissue culture, by using tobacco as a model. In this study, plasmid DNA containing sequences of Cas9, guide RNA, and fluorescent reporter was introduced into pollen using a biolistic delivery system. Based on the transient expression of fluorescent reporters, the Arabidopsis UBQ10 promoter was found to be the most suitable for driving expression of the delivered gene in pollen tubes. We also evaluated delivery efficiency in male germ cells in the pollen by expression of the introduced fluorescent marker. Mutations were detected in the target gene in the genomic DNA extracted from CRISPR/Cas9 introduced pollen tubes but were not detected in the negative control. Bombarded pollen germinated pollen tubes on the stigma and produced two sperm cells within the pistil. We also observed ovules showing fluorescence derived from bombarded pollen. The findings of this study provide important insights into the editing of pollen tube genomes and the delivery of genome-modified male germ cells for seed production.

plant biology↗