FLASH: A high-efficiency soybean-native protoplast platform for real-time protein localization and interaction studies

Soybean (Glycine max (L.) Merr.), is one of the world’s most important oilseed and economic crops, yet its cell-biology-oriented gene-function studies remain hampered by low transformation efficiency and poor detection of fluorescent tags such as GFP. Researchers therefore routinely resort to heterologous systems like Arabidopsis or Nicotiana benthamiana, risking mis-localization and artifactual activity of soybean proteins and compromising physiological relevance. A robust, soybean-based platform is urgently needed. In this study, we introduce FLASH (Fluorescent Localization Assay for Subcellular in Hairy-root-derived callus protoplasts). Transgenic hairy roots expressing fluorescent proteins are bulked into callus, from which protoplasts are isolated in a single step and > 80% of cells display bright, stable fluorescence within 24 h. Using FLASH we monitored, in real time, light-induced assembly of GmCRY photobodies in both the nucleus and cytoplasm, and confirmed nuclear co-localization of GmCRY1b with STF1. FLASH offers a high-fidelity, high-throughput solution for subcellular localization, protein–protein interaction, and phase-separation studies of soybean proteins.