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Identification of unknown cytosolic factors involved in the targeting of organellar proteins in plants

Grant number: 13/24078-9
Support type:Scholarships abroad - Research Internship - Doctorate
Effective date (Start): February 01, 2014
Effective date (End): October 31, 2014
Field of knowledge:Biological Sciences - Genetics - Plant Genetics
Principal Investigator:Márcio de Castro Silva Filho
Grantee:Larissa Spoladore
Supervisor abroad: Jelena Brkljacic
Home Institution: Escola Superior de Agricultura Luiz de Queiroz (ESALQ). Universidade de São Paulo (USP). Piracicaba , SP, Brazil
Local de pesquisa : Ohio State University, Columbus, United States  
Associated to the scholarship:11/13185-3 - Characterization of the role of cytosolic proteins in the localization of organellar proteins in Arabidopsis thaliana., BP.DR

Abstract

The goal of this study is to investigate unknown cytosolic factors involved in the targeting of organellar proteins in plants. Cytosolic proteins that interact with dual-targeted proteins were listed from a protein-protein interaction (PPI) network generated in silico, as candidates to act on regulating protein transport. One of these was GRF9, a protein of the 14-3-3 family (AT2G42590.3). This protein is being characterized, and its participation in sub-cellular targeting is being investigated with experiments such as gene silencing to verify the consequence the absence of GRF9 will have in the correct localization of THI1, a protein targeted to both mitochondria and chloroplasts. If it does not show an association with this function, we planned to investigate other cytosolic proteins found in the PPI network. We believed we needed a more direct and fast plan to lead us to the answers of the project. To obtain the answers to our question more directly, we plan to use forward genetic studies. Since the role of GRF9 on the protein targeting is not yet confirmed, and might not show a correlation, the generation of random mutations on the Arabidopsis thaliana genome, following a phenotypic screening of the resulting lines, can lead to the discovery of mutants which had the transport of proteins to the chloroplasts impaired, possibly by an unknown factor, which can then be mapped and eventually identified. We believe this approach will provide us with excellent answers, and even more than one cytosolic factor can be found in a few months, with extensive screenings at the Ohio State University facilities for growing the plants, and confirmation of their role in sub-cellular protein targeting, as they are mapped and identified, which will need more months. In view of the fact that there are many unanswered questions concerning the chloroplast protein import mechanism, it seems likely that additional proteins, or regulatory factors, remain to be discovered. The availability of the ABRC center for an immense collection of mutants will be excellent for the project advancements, to increase the speed to confirm findings, since these seeds will be readily available for testing, as the genes are discovered with the screening and mapping. Dr. Jelena Brkljacic is the Associate Director of ABRC and has worked in The Ohio State University on plant-specific nuclear envelope targeting and nuclear pore biology, where she gained extensive cell biology expertise. Dr. Brkljacic has accepted the candidate to work in her lab for one year, to provide the tools to conduct the project, and access to the ABRC collection of mutants. The characterization of GRF9 will continue along with the advancements of this new project, to verify its possible role in regulating organellar protein targeting in Arabidopsis. (AU)