ENGINEERED GFP AS A VITAL REPORTER IN PLANTS

Background: The green-fluorescent protein (GFP) of the jellyfish Aequo rea victoria has recently been used as a universal reporter in a broad range of heterologous living cells and organisms. Although successful in some plant transient expression assays based on strong promoters o r high copy number viral vectors, further improvement of expression ef ficiency and fluorescent intensity are required for GFP to be useful a s a marker in intact plants. Here, we report that an extensively modif ied GFP is a versatile and sensitive reporter in a variety of living p lant cells and in transgenic plants. Results: We show that a re-engine ered GFP gene sequence, with the favored codons of highly expressed hu man proteins, gives 20-fold higher GFP expression in maize leaf cells than the original jellyfish GFP sequence. When combined with a mutatio n in the chromophore, the replacement of the serine at position 65 wit h a threonine, the new GFP sequence gives more than 100-fold brighter fluorescent signals upon excitation with 490 nm (blue) light, and swif ter chromophore formation, We also show that this modified GFP has a b road use in various transient expression systems, and allows the easy detection of weak promoter activity, visualization of protein targetin g into the nucleus and various plastids, and analysis of signal transd uction pathways in living single cells and in transgenic plants. Concl usions: The modified GFP is a simple and economical new tool for the d irect visualization of promoter activities with a broad range of stren gth and cell specificity. It can be used to measure dynamic responses of signal transduction pathways, transfection efficiency, and subcellu lar localization of chimeric proteins, and should be suitable for many other applications in genetically modified living cells and tissues o f higher plants. The data also suggest that the codon usage effect mig ht be universal, allowing the design of recombinant proteins with high expression efficiency in evolutionarily distant species such as human s and maize.