Vicki Vance - US grants

The objectives of the proposed research are to understand the interactions which occur in a synergistic viral infection. Proposed experiments focus on the interaction of two unrelated (+)-stranded RNA viruses of plants, potato virus X (PVX) and potato virus Y (PVY). In mixed tobacco infections with these two viruses the accumulation of PVX is greatly increased over that in singly infected plants, while that of PVY is unchanged. The immediate goal of the work is to determine the level at which the increased accumulation of PVX is regulated. The steady state levels, time course of accumulation and rates of synthesis of coat protein and various viral RNAs will be measured in singly and doubly infected plant protoplasts. Later aims are to pinpoint the specific sequences on the PVY and PVX genomes which are important for the increased accumulation of PVX in co- infected cells. These experiments will use transgenic plants expressing subsets of the PVY genome, as well as genetically engineered PVX mutants. Recent studies have shown similarities in diverse plant and animal viruses based on genome organization,mechanisms of gene expression, and sequence homology of virally encoded, nonstructural proteins. These results suggest that elucidation of molecular mechanisms for virus interactions obtained in one system may well apply to a diverse group of other virus systems. This work should give insight into a fundamental biological phenomenon and, particularly if the results are found to apply to a number of viral systems, may be of economic importance.

DESCRIPTION (from the application): Post-transcriptional gene silencing (PTGS) is an ancient eukaryotic regulatory mechanism in which a particular RNA sequence is targeted and destroyed. Although PTGS occurs in diverse organisms, the cellular components of the pathway and its regulation are not well understood. The helper component proteinase (HG-Pro) of plant potyviruses has been shown to suppress PTGS in plants. Using HG-Pro as bait in a yeast two-hybrid system, we have identified a novel calmodulin-related protein (termed rgsCaM) that interacts with this viral suppressor of PTGS. rgs-CaM, like HG-Pro itself, suppresses both initiation and maintenance of PTGS. Our hypothesis is that HC-Pro-suppression of PTGS is mediated by its interaction with rgs-CaM. Here we propose to exploit HC-Pro and rgs-CaM as tools to dissect the mechanism of silencing in plants. The first four aims of the proposal focus on the role of rgs-CaM in PTGS, identifying domains of the protein required to suppress PTGS as well as those involved in the rgs-CaM/HC-Pro interaction. Several approaches will be used to interfere with expression of rgs-CaM and the effect of these manipulations on PTGS and its suppression by HC-Pro will be assayed. The third aim addresses mechanistic questions, determining how and where HC-Pro and rgs-CaM act with regard to known steps in PTGS. The last two aims focus on the role of other plant proteins in the HC-Pro-mediated suppression of PTGS. We will extend investigations of three other HC-Pro-interacting proteins using the approaches that successfully identified rgs-CaM as a regulator of PTGS. Gene array technology will be used to identify genes that are regulated in response to PTGS or its reversal by HC-Pro. Finally, we will exploit the model genetic organism Arabidopsis thalianato screen for mutants that interfere with suppression of PTGS in response to HC-Pro. The plant silencing system serves as a model to understand similar pathways in animals, and has potential to be exploited for gene therapy applications and manipulation of gene expression. Given the antiviral nature of gene silencing in plants, understanding PTGS in plants could well lead to the development of antiviral strategies in humans.

Scientific Objectives and Approaches. Endogenous small regulatory RNAs, including short interfering RNAs (siRNAs) and microRNAs (miRNAs), mediate development and responses to stress in plants as in other eukaryotic organisms. Knowledge of the number, sequence and expression pattern of such small RNAs as well as their regulatory targets is essential in order to understand the basic biology of plants as well as to develop high yielding, stress resistant crops. Experiments are to extensively analyze the sequence and expression patterns of all or nearly all of the small RNAs expressed in two important crop plants, rice and maize. The first aim is to clone and sequence small RNAs isolated from rice and maize (~10,000/organism), providing the first extensive systematic examination of small RNA populations in both species. miRNAs in the small RNA populations of both plants will be identified using both experimental and computational approaches and the number and location of rice miRNA genes annotated. The last two aims focus on understanding the biogenesis and function of the monocot miRNAs, establishing miRNA expression patterns during development and in response to various kinds of stress and mapping the ends of the primary precursor RNAs for rice miRNA genes and a subset of those in maize. A web interface to all data generated in the proposal will be made available to the research community, including a database of all small RNA sequences, predicted miRNA precursors and targets and other associated information through a searchable website.
Broader Impacts. The recent discovery that endogenous siRNAs and miRNAs play important roles in the regulation of eukaryotic gene expression has put small RNA biology at the forefront of experimental science. The work will extend the current database of small RNAs in plants. The project is a three-University collaborative effort that offers unique mentoring opportunities. Two of the participating Universities are EPSCoR institutions in South Carolina: University of South Carolina (USC), with a large female (54%) and minority (22%) enrollment and Claflin University, the oldest historically black University in the state. The third is University of California, Davis (UC Davis). An exchange program will be established between Claflin University, USC and UC Davis, allowing promising young Claflin and USC students to participate in cutting edge research activities at each University.
Specific deliverables to the research community: 1) An extensive database of small RNA sequences and associated information will be made freely available via the project website. 2) A candidate miRNA-target dataset generated by the findMiRNA algorithm that will enable further bioinformatic analysis of miRNAs and their targets. 3) Microarrays of all identified rice miRNAs sequences and all putative maize miRNAs will be made freely available to the community as soon as the methodology is established at a small cost to include producing the array as well as shipping expenses. Distribution of microarrays will be handled by the University of South Carolina Institute for Biological Research and Technology (IBRT).

The project focuses on the role of RAV, a presumptive transcription factor, in mediating the action of a potent viral suppressor of RNA silencing called helper component proteinase (HC-Pro). HC-Pro is a multifunctional viral protein that suppresses RNA silencing and causes anomalies in the biogenesis and function of a number of small regulatory RNAs, including endogenous microRNAs (miRNAs) and the short interfering RNAs (siRNAs) that mediate the RNA silencing process. Preliminary studies indicate that tobacco RAV and the closely related Arabidopsis protein, RAV2, are endogenous regulators of RNA silencing that are required for the for HC-Pro suppression of virus induced silencing but not HC-Pro associated defects in the endogenous small RNA pathways. The first aim of this project addresses the hypothesis that HC-Pro uses RAV2 to regulate the expression of genes required for the suppression of RNA silencing. Genes that are directly regulated by RAV2 will be identified using whole genome tiling arrays and chromatin immunoprecipitation techniques, and the role of these genes in RNA silencing will be assessed by genetic analysis. The second aim of the project is to map the domains of both HC-Pro and RAV2 that are required for their interaction in vivo and in vitro, determine the intracellular localization of RAV2 and whether it is altered in HC-Pro plants. This research is likely to contribute significantly to the understanding of RNA silencing, its interconnection with endogenous small RNA pathways and the mechanisms by which viruses usurp endogenous control systems for counter-defensive purposes.

Outreach: The research will be integrated into the educational system at the University of South Carolina via participation in the University 101 program for first year students, which has received national recognition for excellence, and through summer rotations for undergraduates in research through the auspices of the Undergraduate Research in Integrative Evolutionary Biology (NSF-REU). The educational outreach component of the research plan will emphasize encouragement for female and under-represented minority students and help provide them with an accessible pathway into the research sciences. Once a year the project will participate in a science section of University 101 using the ongoing research as an example of modern scientific inquiry as a means of reaching a broad group of students early in their college experience. In addition, summer internships will include lab work, organized research meetings and a formal presentation of work through the auspices of the Undergraduate Research in Integrative Evolutionary Biology (NSF-REU). Senior personnel on the proposal, as well as the PI, will participate actively in both teaching and mentoring.

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.

Plants and plant-derived products have long been popular complementary health interventions in America and throughout the world. Recently, there is increased evidence that plant RNAs ingested in the course of eating ordinary plant-based foods are taken up by the mammalian digestive tract and remain functional. These observations raise the possibility of incorporating delivery of modern molecular RNA-based therapeutics into what has traditionally been a complementary health intervention modality. MicroRNAs (miRNAs) are a class of small non-coding RNAs that play a critical role in gene expression in eukaryotes, controlling virtually every physiological process in the body. Reduction in certain miRNAs is associated with many diseases, and restoration of the missing miRNA blocks disease progression. Although the therapeutic potential of these miRNAs is clear, delivery of the needed miRNA to the diseased cells is a critical barrier to implementation of the therapy. This R21 proposal is focused on developing a safe, effective, and economically feasible strategy for delivering therapeutic miRNAs in vivo. The strategy is based on the scientific premise that edible plants can be used as biofactories to produce therapeutic mammalian miRNAs that can be administered via ingestion. This strategy represents a fundamental shift from the current mainstream approach in the field, which has focused on synthesizing miRNAs as double-stranded ?miRNA mimics? and delivering them intravenously, formulated with lipids or liposomes. The idea of using plants to produce therapeutic miRNAs is particularly attractive for several reasons. Plants can be bioengineered to make miRNAs of any desired sequence and plant-made miRNAs are biologically natural - not double-stranded or chemically modified. Furthermore, plants package miRNAs into nanoparticles that resemble exosomes. These plant exosomes stabilize miRNAs in the GI tract and are taken up via intestinal stem cells and macrophages, providing an effective delivery mechanism. Finally, the plant exosomal membranes themselves have been shown to have anti- inflammatory properties. The proposed experiments build on our preliminary results from feeding experiments using plant-produced mammalian tumor suppressor miRNAs to reduce tumor burden in the well-established Apcmin/+ mouse model of colon cancer. The goals of the proposal are three-fold: 1) Characterize the miRNA content of exosomes isolated from our transgenic plants that have been bioengineered to produce validated mammalian tumor suppressor miRNAs; 2) establish methods to enhance packaging of bioengineered mammalian miRNAs into the plant exosomal fraction; and 3) test the efficacy of orally administered tumor suppressor miRNA-containing plant exosomes in chemoprevention, using the APCmin/+ mouse model of colon cancer. To our knowledge, we are the only group exploring this potentially transformative approach to enabling the use of miRNA therapeutics.