A high‐throughput transient expression system for rice

• Published in: Plant, cell and environment Vol. 42; no. 7; pp. 2057 - 2064
• Main Authors: Page, Mike T., Parry, Martin A.J., Carmo‐Silva, Elizabete
• Format: Journal Article
• Language: English
• Published: United States Wiley Subscription Services, Inc 01-07-2019; John Wiley and Sons Inc
• Subjects: Calories; carboxysome; Cassettes; cellular localisation; Chloroplasts; Climate change; Cloning; Cloning, Molecular; confocal microscopy; Crop yield; Crops; Cyanobacteria - genetics; Gene expression; Gene Expression Regulation, Plant; Genes, Plant; Genetic transformation; Golden Gate; High-Throughput Screening Assays; high‐throughput; Modular systems; Nutrient deficiency; Oryza; Oryza - genetics; Oryza sativa; Plants, Genetically Modified; Plasmids; Protein interaction; Protoplasts; Rice; synthetic biology; Transformation, Genetic; Transgenes; Transgenic plants; transient expression; Oryza sativa; synthetic biology; transient expression; confocal microscopy; high-throughput; Golden Gate; protoplasts; cellular localisation; Rice; carboxysome
• ISSN: 0140-7791; 1365-3040
• DOI: 10.1111/pce.13542

Rice is an important global crop and represents a vital source of calories for many food insecure regions. Efforts to improve this crop by improving yield, nutritional content, stress tolerance, or resilience to climate change are certain to include biotechnological approaches, which rely on the expression of transgenes in planta. The throughput and cost of currently available transgenic expression systems is frequently incompatible with modern, high‐throughput molecular cloning methods. Here, we present a protocol for isolating high yields of green rice protoplasts and for PEG‐mediated transformation of isolated protoplasts. Factors affecting transformation efficiency were investigated, and the resulting protocol is fast, cheap, robust, high‐throughput, and does not require specialist equipment. When coupled to a high‐throughput modular cloning system such as Golden Gate, this transient expression system provides a valuable resource to help break the “design‐build‐test” bottleneck by permitting the rapid screening of large numbers of transgenic expression cassettes prior to stable plant transformation. We used this system to rapidly assess the expression level, subcellular localisation, and protein aggregation pattern of nine single‐gene expression cassettes, which represent the essential component parts of the β‐cyanobacterial carboxysome.
We provide a protocol for the transient expression of transgenes in rice protoplasts. The method is simple, inexpensive, confers a high efficiency of transformation, and is high‐throughput, thus permitting its use in combination with high‐throughput cloning methods. We demonstrate its utility in assessing the localisation of various cyanobacterial proteins.