A highly efficient rice green tissue protoplast system for transient gene expression and studying light/chloroplast-related processes

Here, we report a simplified method for isolating protoplasts from normally cultivated young rice green tissue without the need for unnecessary chemicals and a vacuum device. Transfections of the generated protoplasts with plasmids of a wide range of sizes (4.5-13 kb) and co-transfections with multiple plasmids achieved impressively high efficiencies and allowed evaluations by 1) protein immunoblotting analysis, 2) subcellular localization assays, and 3) protein-protein interaction analysis by bimolecular fluorescence complementation (BiFC) and firefly luciferase complementation (FLC). Importantly, the rice green tissue protoplasts were photosynthetically active and sensitive to the retrograde plastid signaling inducer norflurazon (NF). Transient expression of the GFP-tagged light-related transcription factor OsGLK1 markedly upregulated transcript levels of the endogeneous photosynthetic genes OsLhcb1, OsLhcp, GADPH and RbcS, which were reduced to some extent by NF treatment in the rice green tissue protoplasts.We show here a simplified and highly efficient transient gene expression system using photosynthetically active rice green tissue protoplasts and its broad applications in protein immunoblot, localization and protein-protein interaction assays. These rice green tissue protoplasts will be particularly useful in studies of light/chloroplast-related processes.Transient expression assays allow rapid and high-throughput analysis of genes in plants [1,2] and thus have become widely used for characterization of gene function. Arabidopsis, maize [3] and tobacco protoplasts [4], tobacco leaf epidermal cells [5], tobacco BY-2 cells [6] and onion epidermal cells [7] are commonly used for transient assays in gene expression, protein subcellular localization, protein-protein interaction and protein activity studies. Accordingly, several methods for transient gene expression have been developed, such as PEG-mediated protoplast transfection [8], biolistic bombardment [9] an