One of the most exciting advances in modern molecular biology was the recent discovery that small RNAs regulate numerous cellular activities including development and virus infection through mechanisms referred to as RNA silencing. RNA silencing is an ancient, widespread mechanism for regulating eukaryotic gene expression, and is also used as a means of artificially reducing gene expression in a wide variety of important plants and animals. Based on very limited findings with plant and animal viruses, host small RNAs can be usurped to aid in virus replication or target viruses for destruction. Despite considerable efforts, little is understood about whether small RNAs derived from viral genomes can also control host gene expression, which could have substantial consequences for disease production. This project will combine the best studied viral replication system, Turnip crinkle virus (TCV)and its satellite RNA satC, with the best developed host silencing model, Arabidopsis thaliana. Preliminary experiments have already demonstrated that host enzymes dice TCV and satC into small RNAs that are mainly derived from "hotspot" regions of their genomes. One TCV small RNA tested was able to reduce the expression of at least six host mRNAs, which was related to symptom production and may be providing a more amenable environment for a successful infection. To gain an understanding of (1) the full range of genes targeted by TCV/satC; (2) how the enzymes that generate the viral small RNAs chose their target sequences, and (3) how the TCV coat protein, which naturally suppresses host RNA silencing, contributes to enhancing TCV replication while suppressing satC replication, this research will identify additional cellular targets of TCV and satC vsRNAs, determine the sequence and structural requirements for viral small RNA excision and analyze if the coat protein is responsible for altering the levels of specific small RNAs. This research will help blend together the currently separate fields of RNA virus replication and RNA silencing, dispel incorrect assumptions about RNA virus replication in plants and provide new ideas for the full role of viral RNA silencing suppressors. Successful completion of these experiments should significantly advance our understanding of the interplay between the host RNA silencing pathway and invading viruses with applications to both animal and plant viral pathogenesis, and will likely provide fundamental new information in the broad area of RNA silencing in plants.
Broader Impact of the Project (education and outreach): Research and knowledge acquisition is being paired with promotion of teacher training in Prince George's County, a substantially minority school district adjacent to Washington DC. An effort was initiated by the PI in 2005 to improve teacher understanding of cutting edge Biology/Virology at DuVal High School. Teachers reported that their students improved both their understanding and enthusiasm for biology and midyear biology assessments improved by 100%. By also working weekly with AP Biology students, all students took the AP exam (compared with only 2 last year) and all applied to and were admitted to research-oriented universities, which were not their original choices (including two students who will join us at the University of Maryland- which is rare for this school district). This pilot program has led to the development of a two week summer workshop to work with 30 high school teachers selected from throughout the county (50 applications were received). The final lecture of the workshop will incorporate the development of the ideas that led to this research project and progress will also be presented at future planned summer workshops. Continued interaction is planned with AP biology students in the Fall, with each session leading off with the "results of the week", which will allow students to follow how this research is planned and how new ideas are generated. By pairing up all 30 teachers at the workshop with other investigators in the College of Chemical and Life Sciences, many with NSF funded research, the aspiration is to expand this model and excite AP-biology students throughout the county by describing how cutting-edge research is conducted and the role that NSF plays in advancing knowledge acquisition and science education.
