Project Summary This project will use model RNA plant viruses to elucidate the mechanisms used by viruses to target nucleoli and regulate chromatin dynamics and host gene expression. Specifically, we will investigate how the p26 protein from Pea enation mosaic virus 2 (PEMV2) modulates host chromatin and gene expression by undergoing liquid-liquid phase separation (LLPS) and partitioning into the nucleolus. We will perform both Assay for Transposase-Accessible Chromatin using sequencing (ATAC-seq) and RNA sequencing (RNA-seq) analyses during infection with wild-type or PEMV2-mutant viruses. These experiments will allow us to determine the extent to which p26 and PEMV2 influence chromatin accessibility and gene expression. The overarching objective of this study is to ascertain whether p26 and PEMV2 repress antiviral genes by inducing chromatin compaction while concurrently enhancing the expression of pro-viral genes by relaxing the surrounding chromatin. Next, we will utilize 2'-O-methylation sequencing (2OMe-seq) to determine whether p26 disrupts the function of fibrillarin in the nucleolus to alter the methylation patterns of small nuclear RNAs (snRNAs). Since disruption of snRNA methylation impacts pre-mRNA splicing, we will investigate whether p26 alters alternative splicing pathways to alter host protein expression and/or function. In addition to the research objectives outlined above, this project places a strong emphasis on training undergraduate researchers to conduct experiments and analyze data using safe and accessible plant virus systems. To further enhance the impact of this work on the undergraduate student body, the research findings will be directly incorporated into the undergraduate bioinformatics course at the University of Missouri-Kansas City (UMKC). This way, >20 students/year will be exposed to hands-on bioinformatics techniques and gain valuable skills in analyzing real-world data. Overall, this study will provide a comprehensive understanding of how nucleoli shape virus-host interactions and provide meaningful research experiences for underrepresented students at UMKC.