An emerging challenge pursuant to acquisition of genomic sequences is the need for efficient tools for specific and regulatable disruption of gene expression to establish gene function. This SGER project aims to use a strategy for targeted and tightly controlled degradation of any cellular RNA in plants, using Arabidopsis as a test organism. The method depends on using RNase P, a ubiquitous enzyme involved in tRNA biogenesis, as a reagent that specifically cleaves an endogenous target RNA and disrupts either its translatability or other biological function(s). This RNase P-mediated cleavage depends on the expression of another small RNA called an external guide sequence (EGS), which when bound to the target mRNA renders the resulting complex a substrate for RNase P. By comparing the efficacy, sequence specificity, inducibility and cell autonomy of the RNase P- and EGS-based method versus RNAi in disrupting the expression of target mRNAs of reporters (luciferase, green fluorescent protein) and tissue-specific transcription factors (WEREWOLF and GLABROUS1), the pros and cons of these potential gene-function discovery tools will be elucidated. In addition to serving as a functional genomics tool for plant biologists, the EGS method could be used to enhance agricultural productivity by engineering disease resistance or altering metabolic/physiological traits. Broader impacts of the proposed research .
The proposed studies are based on a solid foundation of promising preliminary results in a monocot and a dicot. The project will enable (i) multi-disciplinary state-of-the-art training for researchers at various stages in their careers, (ii) a network of academic research collaborations and enhancement of the research infrastructure at a pioneering land-grant institution, and (iii) the development of a new, exciting technology with significant payoffs in agricultural biotechnology.
It is anticipated that after two years all the necessary experimental protocols to help other researchers design their own studies for targeted disruption of gene expression using the EGS method will become available. To ensure that the results of this study will have maximum impact in the research community, reagents and tools needed for the EGS strategy will be provided immediately upon request.
