Using combinatorial control to improve transgene expression specificity in plants

DESCRIPTION (provided by applicant): It is currently estimated that 15% of the world's population is undernourished and 5 million childhood deaths a year are attributable to malnutrition. The ability to feed the world in the future will be even more difficult due to population growth, climate change, water scarcity, and competition for land. It is widely recognized that advances in agricultural biotechnology will be required to meet the world's future nutritional needs. In nature, gene expression is regulated at multiple levels including transcription, alternative splicing, transcript stability, and translation. In contrast, the agricutural biotechnology industry has relied primarily on constitutive transcriptional control to regulate the expression of traits introduced into crops. This has been sufficient for the relatively simple trais that have been commercialized to date, but the complex traits that will be needed to meet future food demands are expected to require increased expression specificity. Thelong term goal of this project is to assemble a portfolio of modular expression elements that can be deployed in a combinatorial fashion to provide predictable and tunable expression solutions to the agricultural biotechnology industry. The goal of the Phase I component of this project is to demonstrate proof-of-concept that heterologous miRNA binding elements and pre-mRNA splicing elements can be used to increase the expression specificity of transcriptional promoters in the model plant Arabidopsis. As a specific product concept, Phase I research will focus on designing a root stele-specific expression cassette useful for regulating traits that target soybean cyst nematode. Our specific aims are to: 1) validate a modular post-transcriptional regulatory element that enhances stele expression specificity, and 2) create stele-specific expression cassettes that are combinatorially controlled by transcriptional promoters and post-transcriptional regulatory elements. Selection of miRNA binding elements will be based on recently published data demonstrating cell type-specific expression patterns of specific miRNAs that are inversely correlated with the expression patterns of their respective targets. Alternative splicing events wil be identified through bioinformatic analysis of cell type-specific RNA-seq and microarray expression data. Identified posttranscriptional regulatory elements will be validated in the context of a constitutive promoter and then tested for their ability to increase the expression specificityof a stele-enriched promoter. After demonstrating proof-of-concept of combinatorial control, Phase II research will expand this approach to crops including economically important cereals like corn. PUBLIC HEALTH RELEVANCE: It is currently estimated that 15% of the world's population is undernourished and 5 million childhood deaths a year are attributable to malnutrition. The ability to feed the world in the future will be even more difficult due to population growth, climate change, water scarcity, and competition for land. It is widely acknowledged by experts that increased agronomic productivity through biotechnology will be needed to meet future food demands. The goal of this project is to facilitate biotechnology approaches that lead to the production of more crops on less land with fewer resources.