<scp>RNAi</scp> reveals a unique set of kinesins mediating chloroplast motility in the giant cytoplasm of <i>Bryopsis</i> (Ulvophyceae), 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 implemented RNAi in Bryopsis, a green feather alga that develops a coenocytic thallus >10 cm in length without cytokinesis. In vitro-transcribed double-stranded RNA (dsRNA) was either mixed with extruded cytoplasm in the presence of polyethylene glycol and then regenerated into thalli or directly introduced into the cytoplasm by perfusion. Within several days, target-gene transcript levels decreased, and the expected phenotypes emerged, indicating effective RNAi. We designed dsRNAs for all 34 kinesin superfamily genes of a model Bryopsis strain, delivered them by both methods, and monitored chloroplast distribution and motility by microscopy. Knockdown of KCBP-type kinesin-14VI (Kin14VIa and Kin14VIb), which drive retrograde chloroplast transport in the moss Physcomitrium patens, and of the apparently Bryopsis-specific kinesin-14II (Kin14IIb) suppressed retrograde motility, resulting in apical chloroplast accumulation. In addition, RNAi of the Bryopsidales-specific kinesin-14VI (Kin14VIc) reduced both retrograde and anterograde movements, implying that multiple Kin14 motors contribute to chloroplast transport in Bryopsis. In contrast, knockdown of Kin12c, a member of the kinesin-12 family that is known to be essential for cytokinesis in land plants, caused basal chloroplast enrichment, indicating divergent evolution of this motor family in the green lineage. Together, these results establish RNAi as a robust loss-of-function approach in a coenocytic alga and reveal lineage-specific kinesins as key drivers of chloroplast motility within its giant cytoplasm.