This award is funded under the American Recovery and Reinvestment Act of 2009 (Public Law 111-5).
Plant pathogens (viral, fungal, bacterial, or pest) affect a significant number of food crops worldwide, often indiscriminately. The impact is considerable and far-reaching, particularly as these agents have numerous invasion strategies. One important plant defense mechanism employs ribosome-inactivating proteins (RIPs), which are naturally occurring cytotoxic agents. Their biochemical activity is to depurinate the universally conserved sarcin/ricin loop (SRL) of rRNA, resulting in the cessation of protein synthesis in the infected cells. Pokeweed Antiviral Protein (PAP) is a type I RIP with broad-spectrum antiviral activity against plant and animal viruses. In addition to the canonical rRNA depurination activity, PAP also possesses mRNA recognition, cap binding, and depurination activities that are distinct from other RIPs. Thus, PAP has demonstrated dual access to the translation machinery and is a model for agents that disrupt eukaryotic translation. The goal of this project is to characterize the mechanism by which PAP selects RNA targets for depurination. The PI will employ a combination of biophysical and biochemical approaches to (1) determine the extent and affinity of eukaryotic initiation factor (eIF) participation in selection of RNA and (2) identify the essential mRNA structural elements that affect PAP affinity and enzymatic activity. Oligonucleotides of capped and uncapped mRNA with differing secondary structure will be used in this study to elucidate these structural requirements. This research will increase our understanding of viral infections and how they affect protein synthesis, potentially leading to new anti-viral approaches. Such insight into plant defense mechanisms will guide practical strategies to reduce crop losses due to pathogen infection.
Broader Impacts: This research will be performed primarily by undergraduate science majors at John Jay College of the City University of New York (CUNY). CUNY is the nation's largest urban public university system, serving over 450,000 students. Notably, the training will be conducted on a campus that enrolls more than 60% Hispanic and African American students and is the largest Hispanic-Serving Institution in the northeastern U.S. and the fourth largest in the nation. During her first five years in the Department of Sciences, the PI has trained a total of 22 students in her research laboratory; this number includes 12 undergraduates. The majority of these students have been of a minority demographic and all but two have been women. The educational impact of this project includes training in modern biophysical and biochemical techniques that are an extension of laboratory classroom skills. The research includes approaches that are suitable for the involvement of students at many levels (high school, undergraduate, graduate, and post-graduate), thus providing comprehensive training while producing scientifically literate citizens. Upon completion of their training, students will be well-equipped for advanced degree programs and/or careers in the STEM fields. The research relies heavily on peers as co-researchers in its approach; thus cooperativity and teamwork skills are naturally incorporated into the students' training. In addition to providing research opportunities for undergraduates, this project allows participating students to travel annually to a national conference. This will give them an opportunity to interact with the broader scientific community, to present experimental results to other researchers, to network with peers from other institutions, and meet with prospective graduate school representatives. Through this project, the students will learn how high quality basic research contributes to larger areas of science and society such as improved agricultural practices, more robust agricultural crops, secure food supply protection, and the nation's economy.
