A universal spikey silica nanoparticle-mediated siRNA delivery for red seaweeds and land plants reveals PyKNOX ‘s role during haploid-diploid transition in Pyropia yezoensis

Delivering siRNA into cells for targeted gene silencing provides an efficient way to analyze genes’ biological function. In some plants and seaweeds, especially those with important economic values, such as land plants apple and hydrangea, red seaweed Pyropia yezoensis, low transformation efficiency or low regeneration rates strongly limited our study in elucidating molecular mechanisms in the growth and development of these species. Here, we prepared a spikey silica nanoparticle (SSN) modified with an amino-group, nanoparticle SSN-NH₂, which efficiently delivered siRNA into conchospores and archeospores of P. yezoensis and successfully induced gene silencing. Furthermore, our findings indicate that this method is similarly effective in the protoplasts of various land plants, such as tobacco, apple, and hydrangea. Using this approach, we investigated the function of a KNOTTED-like homeobox gene (PyKNOX) in life cycle regulation. We found that PyKNOX is transcriptionally active in both conchosporangia and newly released conchospores. Transient silencing of PyKNOX delayed the entry of conchospores into meiosis, indicating its essential role in meiosis initiation, mediating the fundamental process of diploid-haploid transition in sexual reproduction of P. yezoensis. This study presents the first universal siRNA transfection method applicable to both red seaweed P. yezoensis and land plants, offering a practical and efficient approach for gene function and gene editing studies. Furthermore, our findings highlight the evolutionary conservation of the regulatory mechanisms governing the diploid-haploid transition in sexually reproducing eukaryotes.