Post-translational Modifications as Modulators of Crop Plant Defense Signaling: a Systems Approach

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PI: Jean T. Greenberg (University of Chicago)CoPIs: Stephen J. Kron (University of Chicago) and Howard C. Hang (Rockefeller University)The interaction of pathogenic microbes with plants elicits signaling pathways that can dictate the outcome of infection. Post-translational modifications (PTMs) elaborated after microbial perception enable a flexible mechanism for plants to activate networks of signaling events that promote host resistance. However, proteins such as acetyltransferase (AC-TR) effectors that are injected into plants by microbes can modify host proteins, which are then affected in their signaling functions due to alterations in folding, cellular location, enzymatic activity, ability to bind other proteins and/or stability. The recent availability of genomic sequence data of economically important plants and new technologies now makes it feasible to do systems-level global analysis of PTMs and to further dissect their impact on disease resistance. The goal of this project is to dissect how PTMs of plant proteins during infection modulates the ability of crop plants to defend against pathogenic bacteria. The project will (1) optimize in vitro methods for studying protein acetylation using AC-TR effectors from bacteria and host substrates; (2) perform global analyses of host protein acetylation in tomato using AC-TR effectors and test the role of select acetylated targets in plant defense; and (3) evaluate the impact of acetylation on defense signaling by analyzing global changes host protein phosphorylation, another PTM that is potentially competitive with acetylation. The project will develop reagents that will be used (and disseminated to the community) to quantitatively study signaling dynamics and relate changes in dynamics due to acetylation to infection outcomes. The global analysis of PTMs performed during this research will enhance the value of genomic studies and give a systems-level understanding of defense signaling that will expand the opportunities for crop improvements. The project will integrate research and teaching to provide interdisciplinary training to project personnel in proteomics, chemical biology, biochemistry, molecular genetics and plant-microbe interactions. It will also have broad impact by providing new tools and reagents to the community for analyzing defense signaling, and giving access to students from other universities to practical training through workshops on the analysis of PTMs. Inclusion of students from underrepresented groups will also have an impact on broadening participation in research of diverse groups. A major innovation of the project is the development and application of new methods for analyzing different acetylation sites in proteins at a global scale in relation to other potentially competitive modifications. The approach will be transformative for the study of acetylation in relation to other protein modifications in crop plants and applicable to a broad variety of plants and other organisms. A key outreach component will be three-day workshops for students from other institutions to learn the principles of detecting modifications of plant proteins. Students will gain hands-on experience on the generation and analysis of data on post-translational modifications using mass spectrometry. Additional outreach efforts will include participation in discussions about this project and demonstrations with school groups that visit the University of Chicago and Rockefeller University, coordination with teachers to modernize science curricula and discussions about research with the public at community fairs. This project will provide important interdisciplinary training in chemical biology, proteomics, biochemistry, molecular genetics and plant pathology to project personnel, including postdoctoral scholars and students at the graduate, post-baccalaureate, undergraduate and high school levels. Project personnel will be drawn from diverse groups to broaden participation in research science. Finally, the project will develop useful reagents (antibodies) and protocols to detect modifications in tomato proteins. The public will have access to the information generated by the project such as specific proteins that are subject to modification and approaches to monitor modifications through peer-reviewed publications and the project website. The public will also have access to reagents generated such as antibodies and tomato seeds as well as primary MS/MS spectra upon request. Proteomics datasets will also be deposited in one or more databases such as P3DB (http://p3db.org/).