Ochiai, K. K. | bioRxiv Science

bioRxiv2025

RNAi reveals a unique kinesin mediating chloroplast motility in the giant cytoplasm of Bryopsis, a coenocytic green alga

RNA interference (RNAi) is a powerful tool for protein knockdown and is widely used in model animals and plants. Here, we applied this technique to Bryopsis, the green feather alga that develops a >10 cm coenocytic body in the wild and in laboratory culture. We mixed in vitro-transcribed double-stranded RNA (dsRNA) with extruded cytoplasm in the presence of polyethylene glycol or injected it directly into the cytoplasm, followed by thallus regeneration. After several days, we observed a reduction in the target gene transcript as well as expected phenotypes, indicating the effectiveness of RNAi. We prepared dsRNAs for the sole myosin and all 34 kinesin genes of the model Bryopsis strain, and performed RNAi and time-lapse microscopy to trace chloroplast movement. In addition to KCBP-type kinesins known to drive retrograde chloroplast transport in land plants, RNAi of a Bryopsidales-specific kinesin-14 (Kin14VIc) almost completely suppressed chloroplast motility. Cytoplasmic microtubules remained broadly aligned parallel to the main axis of the thallus following Kin14VIc RNAi. Purified Kin14VIc motor protein showed microtubule-gliding activity and, when artificially tetramerised, processive motility in vitro ([~]250 nm/s), similar to plant KCBP. Thus, this study introduces a powerful gene loss-of-function tool in a coenocytic organism and identifies a uniquely evolved kinesin as a critical driver of chloroplast motility in the giant cytoplasm.

cell biology↗

bioRxiv2023

Genome sequence and cell biological toolbox of the highly regenerative, coenocytic green feather alga Bryopsis

Green feather algae (Bryopsidales) undergo a unique life cycle in which a single cell repeatedly executes nuclear division without cytokinesis, resulting in the development of a thallus (> 100 mm) with characteristic morphology called coenocyte. Bryopsis is a representative coenocytic alga that has exceptionally high regeneration ability: extruded cytoplasm aggregates rapidly in seawater, leading to the formation of protoplasts. However, the genetic basis of the unique cell biology of Bryopsis remains poorly understood. Here, we present a high-quality assembly and annotation of the nuclear genome of Bryopsis sp. (90.7 Mbp, 27 contigs, N50 = 6.7 Mbp, 14,034 protein-coding genes). Comparative genomic analyses indicate that the genes encoding BPL-1/Bryohealin, the aggregation-promoting lectin, are heavily duplicated in Bryopsis, whereas homologous genes are absent in other Ulvophycean algae, suggesting the basis of regeneration capability of Bryopsis. Bryopsis sp. possesses >30 kinesins but only a single myosin, which differs from other green algae that have multiple types of myosin genes. Consistent with this biased motor toolkit, we observed that the bidirectional motility of chloroplasts in the cytoplasm was dependent on microtubules but not actin in Bryopsis sp. Unexpectedly, most genes required for cytokinesis in plants are present in Bryopsis, including those in the SNARE or kinesin superfamily. Nevertheless, a kinesin crucial for cytokinesis initiation in plants (NACK/Kinesin-7II) is hardly expressed in the coenocytic part of the thallus, possibly underlying the lack of cytokinesis in this portion. The present genome sequence lays the foundation for experimental biology in coenocytic macroalgae. Significance statementThe exceptionally coenocytic body and remarkable regeneration ability of Bryopsis have attracted biologists for years. However, molecular biological tools remain underdeveloped, partly due to the lack of genome information. Here, we report high-quality assembly and annotation of the genome, providing a crucial resource for experimental biology and genomics studies of Bryopsis. Furthermore, comparative genomic analysis reveals a unique gene repertoire that possibly underlies the highly regenerative coenocytic body.

plant biology↗