Intellectual Merit: The long term goal of this research is to understand plant anti-pathogen strategies. Ribosome inactivating proteins (RIPs) are linked to plant defense and are found in numerous plant, fungal, and bacterial species. They have a broad spectrum of anti-pathogen properties including antiviral activity. Pokeweed antiviral protein (PAP), found in American pokeweed, is a type I RIP and inhibits protein synthesis by depurinating the sarcin/ricin loop of the large ribosomal RNA. Recent evidence shows that PAP can also depurinate mRNA, bind to m7G caps of mRNA, and interact with translation initiation factors. PAP is expressed in different isoforms and in different compartments of the plant throughout the pokeweed. Formation of homo- and/or hetero-dimers under some conditions has prompted the oligomerization is influenced exposure to various pathogens or stressors. The functional consequences of PAP interactions, isomers, and polymers are not well-understood. This RAPID research project will characterize the various isoforms and oligomeric forms of PAP(1)at various stages in the plants growth cycle, from various tissues/compartments of the plant (e.g. leaves, roots, stems, berries), and(2)in response to exposure to various pathogens and/or stressors. This is a time-sensitive project restricted by the life cycle of the plant and by an agreement with the New York City Central Park Conservancy to harvest pokeweed plant tissues during the upcoming 2010 growing season.
Broader Impacts: This project, which will be carried out primarily by students, includes a summer research training opportunity for students from John Jay College, CUNY to work in the laboratory of Dr. Jorge Vivanco at Colorado State University. The opportunity to participate in the summer experience will provide students with excellent training in multiple biochemical, biophysical, and plant biology techniques. Also, because John Jay College is a primarily undergraduate institution with a population that is largely minority and female, this RAPID activity will increase the likelihood that these underrepresented individuals will pursue postgraduate degrees and continue in science as independent investigators. Moreover, this project will help forge a robust collaboration for future work between the Friedland and Vivanco laboratories and their students. Through collaboration, the scientific impact extend from current knowledge of RIPs to a deeper quantitative understanding and to the plant rhizosphere biology field.
??Plants have developed natural defense mechanisms as ways of protecting themselves against viral agents, bacteria, and fungi. A commonly found defense mechanism involves ribosome-inactivating proteins. Pokeweed antiviral protein is a type I ribosome-inactivating protein loacted in leaves, root, and seeds of Phytolacca americana which is a commonly found weed in North America. The pokeweed plant produces many different types of pokeweed antivitral protein (PAP I, PAP II, PAP-S) at various stages in plant's growth cycle, from various tissues of the plant, and in response to exposure to external stress (such as plant pathogens). The project was designed to characterize the pokeweed antiviral protein expression pattern under normal and stress conditions. PAP possesses two distinct activities: 1) the binding activity to the RNA structure and 2) the depurination of RNA. We have examined PAPs extracted from leaves during two consecutive summers (JJ5 and JJ8), as well as PAP-S purified from the pokeweed berries for these two activities. PAP-S revealed the greatest affinity for the RNA cap structure. JJ5 PAP-I had the least ability to depurinate Tobacco Etch Virus (TEV) RNA. PAP-II from JJ8 showed the greatest depurination ability. PAP-II was shown to have the greatest activity towards viral RNA. This observation suggests that plant leaves which had been exposed to increased pathogenic contact during a particular year, will produce a stronger antiviral protective agent. Students also characterized pokeweed antiviral protein expression in response to two different pathogens (a human pathogen, PA01, and a plant pathogen, DC3000) at Professor Vivanco's Horticulture laboratory, located at Colorado State University during summer of 2010 and 2011. We found pokeweed leaves that had been infiltrated with DC3000 showed apoptotic symptoms without increasing PAP expression. Pokeweed leaves infiltrated with PA01 showed no cell death with "up-regulation" of PAP expression. This indicates that pokeweed defends itself against bacterial invasion by increasing its expression of PAP.
