In plants, RNA silencing is an important adaptive defense against viruses. The several silencing pathways in plants include post-transcriptional gene silencing (PTGS) mediated by small interfering RNAs (siRNAs), regulation of gene expression by microRNAs (miRNA), and siRNA-directed methylation of DNA and associated histones leading to transcriptional gene silencing (TGS) and the establishment of heterochromatin. Most plant viruses have RNA genomes and are targeted by PTGS, which results in degradation of viral genomes and mRNAs. The transcripts of DNA viruses (e.g. geminiviruses) are also subject to PTGS. In addition, because geminiviruses replicate in the nucleus through dsDNA intermediates that associate with cellular histones, their genomes are subject to methylation and TGS. This project is focused on geminivirus chromatin methylation. The studies proposed are based on several recent discoveries from the Bisaro lab which implicate geminivirus genome methylation as an important host defense and suggest that geminiviruses encode proteins that counter this defense by inhibiting methylation. First, using a combination of genetic and biochemical methods, the antiviral nature of methylation will be confirmed. Second, mechanisms by which the related geminivirus proteins AL2 and L2 suppress and reverse TGS will be studied, and the ability of these proteins to alter cytosine and histone methylation status examined. Finally, because geminiviruses offer a unique access to RNA-directed methylation, geminiviruses will be used as model systems to identify novel cellular genes that participate in the methylation pathway. Methylation controls cellular gene expression during normal development and leads to the establishment of heterochromatin. It also maintains genome integrity by silencing endogenous invasive DNAs such as repeated sequences and transposons. Thus studying geminivirus chromatin methylation promises to increase our understanding of viral pathogenesis and a fundamental process that governs the organization and activity of cellular chromatin.
Non-technical Summary In plants, RNA silencing is an important antiviral defense that results in specific degradation of viral mRNA. In the case of DNA viruses, a related process (RNA-directed methylation) leads to the methylation of viral DNA and associated histone proteins that together constitute the viral chromosome. In this project, the role of RNA-directed methylation as a defense against DNA-containing geminiviruses will be determined. The ability of viral proteins to counter this defense and inhibit methylation will also be examined. Finally, geminiviruses will be used as model systems to identify new host genes involved in methylation. Methylation controls cellular gene expression during normal development and is important for organizing inactive regions of cellular chromosomes. Thus studying geminivirus chromatin methylation promises to increase our understanding of viral pathogenesis and a fundamental process that governs the activity of cellular chromatin. A better understanding of host antiviral defenses and viral counterdefense will eventually lead to the development of plants that are better able to withstand or resist virus attack. Further, what is learned in plant systems may also prove applicable to mammalian virus-host interactions. Additional benefits of this project include the training of undergraduate students, graduate students, and postdoctoral fellows (including under-represented minorities) in the concepts and methods of modern molecular biology and virology.
Regulation of gene expression lies at the core of all cellular processes, and it is only recently that we have come to appreciate the importance of epigenetic phenomena as gene regulatory mechanisms. Epigenetic events include chemical modifications to DNA and associated histone proteins that modulate the accessibility of DNA to RNA polymerase II, which transcribes genes to produce mRNA, the first step in gene expression. As a result of work supported by this project, we discovered that plant cells employ repressive DNA and histone methylation as an epigenetic antiviral defense to compact viral chromatin and silence the transcription of geminivirus genes (transcriptional gene silencing, TGS). We also demonstrated that viruses counter TGS by producing proteins that inhibit methylation. Thus, we significantly advanced the field by revealing a new defense-counterdefense network that modulates the pathogenicity of plant DNA viruses, and likely also mammalian DNA viruses as well (e.g. Herpes simplex virus). This information may be valuable for devising new strategies to inhibit DNA virus replication. Of equal significance, we also demonstrated that viruses are effective models for the study of cellular chromatin methylation pathways by using the geminivirus system to identify a new component (an RNA binding protein called DRB3) in the plant DNA methylation pathway. Further, while it is known that repressive methylation of chromatin is accomplished by small RNA-based mechanisms, little is known about how sequences are targeted for methylation. Geminivirus model systems may prove to be important tools for addressing this and other fundamental questions relevant to the regulation of gene activity. Already, we have learned that plant RNA polymerases IV and V play critical but non-essential roles in this process by transcribing DNA targeted for methylation. This project also contributed substantially to the training of a postdoctoral associate, 7 graduate students (1 an under-represented minority), a visiting graduate student (from Pakistan), 8 undergraduate students (one an under-represented minority), and visiting postdoctoral scholars from China, Pakistan, and South Korea. In addition, one minority graduate student participated in an NSF REU experience in the lab (the REU program is sponsored by the Departments of Molecular Genetics and Biochemistry at The Ohio State University). As Director of the interdisciplinary graduate program in Molecular, Cellular, and Developmental Biology at Ohio State (~130 PhD students, ~170 faculty), the PI advises all first year students and, in addition to regular teaching duties, voluntarily participates in a team-taught first-year mentoring course that includes training in the ethical conduct of research.
