This project is part of a long-term effort to understand the mechanism of action of a plant viral protein, helper component proteinase (HC-Pro), which is a potent suppressor of RNA silencing in plants. RNA silencing is used by plants and animals to defend against viral infection and to control expression of the organism's own genes. Therefore, HC-Pro not only blocks anti-viral defense, but also interferes with an important genetic regulatory mechanism in the host plant.
The goal of this project is to determine the role of host plant proteins in HC-Pro function, using genetic, biochemical, and molecular approaches. The project focuses on a novel calmodulin-like protein that interacts with HC-Pro and is itself a suppressor of silencing when over-expressed in plants. Because calmodulin and related proteins bind calcium, which is used as a signal in many biological processes, the work is poised to provide new insights into the role of calcium signaling in the regulation of silencing as well as to contribute significantly to understanding the mechanisms by which viruses usurp endogenous control systems for counter-defensive purposes. The outcomes of this project should have a major impact on the field because investigation into the role of host proteins in viral suppression of silencing has been largely neglected and the role of calcium signaling has not been studied at all. The research should also contribute significantly to advancements in antiviral defense strategies and in biotechnologies that make use of plants. The project will provide summer internships for two students to participate in the research and will emphasize participation of female and minority students. Senior project personnel will participate actively in both teaching and mentoring these interns to help provide them with an accessible pathway into the research sciences.
Project Outcomes Intellectual Merit: How viruses cause disease is an issue that impacts virtually all living organisms. In plants, a pathway called RNA silencing is the major anti-viral defense mechanism. The silencing pathway is triggered by double stranded RNAs (dsRNAs) that arise during viral infection. These dsRNAs are chopped into small RNAs that are then used to specifically target and destroy any other viral RNAs. As a counter-defense, most plant viruses encode proteins that block silencing at some point in the pathway. This NSF project is part of our long-term effort to understand the mechanism of action of one such viral suppressor of RNA silencing called helper component proteinase (HC-Pro). HC-Pro is a multifunctional viral protein that not only blocks RNA silencing, but also causes anomalies in the biogenesis and function of endogenous small regulatory RNAs called microRNAs (miRNAs) that are involved in normal plant development. The goal of this project was to investigate the role of host proteins in various aspects of HC-Pro function with a focus on identifying proteins that control the RNA silencing pathway and/or HC-Pro-mediated suppression of that pathway. Initial work examined the role of CML38, a novel calmodulin-like protein that interacts physically with HC-Pro and is itself a suppressor of silencing when over-expressed in plants. Calmodulins are signaling proteins that bind calcium, which causes a conformational change that exposes hydrophobic domains. Once activated by calcium, they bind to target proteins, usually inhibiting their function. The CML38 experiments focused on determining if HC-Pro functions via its interaction with CML38. Our approach took advantage of the model genetic plant Arabidopsis thaliana. We identified an Arabidopsis plant line carrying a null mutation in the CML38 gene, and used it to examine whether the CML38 protein is required for any aspect of HC-Pro function. We found that CML38 is required for HC-Pro suppression of RNA silencing, and that the requirement is very stringent: two intact copies of the CML38 gene are needed. Interestingly, the developmental anomalies characteristic of plants expressing HC-Pro also required two intact copies of the CML38 gene, but the defects in the endogenous miRNA pathway were independent of CML38. These results identify CML38 as a crucial control point in HC-Pro-mediated suppression of silencing, and raise the possibility that endogenous calcium signaling networks are involved in the regulation of the RNA silencing pathway in plants. Additional experiments further delineated the role of CML38 in HC-Pro suppression of silencing using both in vitro and in vivo approaches. This part of the project explored the role of SBP1, an E3-ubiquitin ligase that interacts with CML38, and addressed the hypothesis that suppression of silencing by HC-Pro and CML38 is mediated via the ubiquitin pathway. We showed that SBP1 is itself a weak suppressor of silencing when over-expressed in plants and that, along with CML38, it plays a role in HC-Pro suppression of silencing. Altogether, the results of this project are a step forward in piecing together the pathway involved in HC-Pro suppression of silencing. The research has provided new insights into the role of calcium signaling in the regulation of silencing, and has contributed to understanding the mechanisms by which viruses usurp endogenous control systems for counter-defensive purposes. Broader impacts: Our long term goal is to help provide undergraduate students from groups that are under-represented in science with an accessible pathway into the research sciences. Our strategy is to integrate research and education by providing research opportunities for these students under the auspices of our NSF projects. We emphasize recruitment of female and minority undergraduates to do research in our lab, and we have been active in providing research opportunities for under-represented groups on all of our NSF-funded projects. This NSF project provided research training and experience for six undergraduate students. Four of these students were women, and two of the women continued their research experience in our lab for two years. Two of the women plan to go into teaching, one is preparing for a career as a medical or crime lab technician, and the fourth entered graduate school in a non-science field. Of the male undergraduate students we trained, one is currently applying to medical school and the other is still at this University.
