Steve Lommel

Agriculture built North Carolina, and it remains our top economic driver. The Golden LEAF Foundation and NC State University have an unprecedented opportunity to establish our state as the global leader in plant sciences ������������������ creating the science and innovation that will transform societal challenges into opportunities for North Carolina agriculture and rural communities. The North Carolina Plant Sciences Initiative (PSI) is a visionary, high-impact venture that will increase agricultural productivity; generate new, value-added agricultural businesses; fuel rural economies; and drive job creation and rural workforce development. PSI is a once-in-a-generation opportunity to pursue our common mission of empowering rural and agricultural communities through economic advancement. No other place in the world has North Carolina������������������s agbioscience assets and agricultural roots. We are uniquely positioned to lead the world in plant science innovation

Activities of Phase 2 include advance planning, schematic design, design development and program development. These will be informed by three task forces comprised of both univeristy faculty leadership and stakeholders addressing governence, scientific focus and advocacy. The outcomes and recommendations from the task forces will drive the decisions of a central planning committee. The NC Agricultural Foundation, Inc. has committed seed funding for a Launch Director, who will lead the planning process, coordinate interdisciplinary faculty intiatitives and help attract new faculty expertise. The completion of Phase 2 will give us the information and working documents we need to construct the Plant Sciences Research Complex and define the scientific leadership required to launch the Plant Sciences Initiative in Phase 3. We anticipate that Phase 2 will begin in Fall 2015 and be completed in Fall 2017. The target for completing construction and full launch of the Plant Sciences Initiative is 2020.

The Agricultural Education Program in the Department of Agricultural and Human Sciences at North Carolina State University will provide the sponsor with curriculum and assessment support for NOrth Carolina's secondary program in agricultural education.

IDEA: Develop and validate a biodegradable cellulose matrix platform technology for seed treatment with active ingredients for crop protection enabled by plant viral nanoparticle and traditional cellulosic pulping processes. Phase 1 of this project demonstrated proof-of-concept for this platform by both demonstrating nematode control efficacy in the tomato test system in growth chamber experiments and establishing a banana pulping strategy to create paper matrices. We also established scientific connections with host communities in sub-Sahran Africa insuring translational implementation of the prototype concept during Phase 2 for abating nematode stresses in subsistence farming that reduce crop yield and quality. GOALS: Our immediate follow-up is to foster the translation of the seed treatment technology by implementing the identified infrastructure for adaption by host communities. This includes workshops with local collaborators demonstrating how the simple act of tearing a section of the active paper, encasing the seed by wrapping, and planting will protect the seed throughout germination. We will also build on our success in pulping banana fibers in Rwanda to implement a low-cost production scheme with locally-relevant materials. This will be done by university-exchange and extension services. As a land-grant university, this know-how of translating high-end technology to developing countries is strongly encouraged and supported, if not expected.

Plant-parasitic nematodes are the most damaging pathogens of soybean in the world. The soybean cyst nematode (SCN; Heterodera glycines) for example infests at least 60% of the soybean acreage in North Carolina and accounts for yield losses in this state of 4 to 8% on an annual basis. This proposal will survey field isolates and greenhouse maintained biotypes of SCN for the presence of viral genomes. Differences between virus-infected and non-infected biotypes will be assayed by several criteria such as infectivity and fecundity. Virus extracts will then be produced from infected SCN populations and attempts will be made to infect virus free isolates of SCN. It is proposed that this study will lead to the development of a viral-based bio-control strategy against SCN.

ARI-R2: Renovation of the North Carolina State University Phytotron for Improved Environmental Control and BSL-3 Containment. North Carolina State University (NCSU) faculty and students have a compelling need for a state-of-the art controlled, environmental facility (Phytotron) with containment for investigation of high-risk plant pathogens and other microbes. The four-story Phytotron was built in 1968 with most of the construction (~$1.5M) and first eight years of operating funds provided by the NSF, and 33 years of continued maintenance and staffing provided by NCSU. After 41 years however, the NCSU Phytotron is in dire need of upgrades to meet present and growing demands for controlled environmental research facilities for plants, insects and small mammals. This application proposes critical infrastructural upgrades to (1) renovate the core environmental system by replacing chillers and pumps, connecting to university chilled water lines, re-insulating chilled water and glycol lines, and applying epoxy coating to chamber and greenhouse floors; (2) replace controllers that modulate the environmental conditions for individual environmental chambers, and modernize and increase the precision of CO2 controls; and (3) convert a plant dark room and greenhouse into a BLS-3 facility for investigations with highest risk viral, bacterial, fungal and nematode plant pathogens and select agents. Intellectual Merit. Biologists in the post genome era face a major challenge in determining the function of the thousands of identified genes, many of which are under environmental control. Often, the phenotype of a mutant and the expression pattern of responsible gene(s) can be properly defined only in precise environmental conditions. Renovation of the Phytotron will provide researchers with the large-scale controlled environment capacity to take full advantage of publically accessible genetic tools not only to accelerate information-rich phenotyping in model plants but to translate the same high-throughput screens to crops, wild relatives and non-native species. Control over CO2 concentration will open new opportunities for NCSU researchers involved in global change research and predicting the responses of individual organisms and ecosystems to environmental change. These demands, as well as the growing need for studies of phenotypic plasticity needing reproducible combinations of climatic factors and ecological studies that require the capacity to simulate a variety of environments, make this facility an essential resource for future, competitively funded investigations. Similarly, upgrading a designated area of the facility for BSL-3 containment will provide a research environment and foster international collaborations to better understand the fundamental biology of plant pathogens that already cause severe disease problems in subtropical/tropical regions and pose an increasing threat to the U. S. and global food supply chain. Broader Impacts. The NCSU Phytotron is an outstanding testament to the value of investing in research infrastructure. This NSF-funded legacy is a valuable University-wide facility and focal point for training and research at all levels. The infrastructure renovations proposed here will allow this unprecedented resource to continue to fulfill this mission by becoming a high-throughput public facility for phenotypic analyses of many different plants. Graduate and undergraduate students from NCSU, summer REU programs involving students from non-Research I universities and other outreach programs for underrepresented groups use the Phytotron for independent research projects under the direction of a diverse representation of faculty who serve as role models and mentors. The renovations will have immediate scientific impacts as NCSU researchers can acquire pathogens and microbes from collaborators around the world by having a centralized facility inspected and known to meet or exceed containment guidelines. Unemployment in the State of North Carolina is over 11% and above the national average.

Develop and validate a biodegradable cellulose matrix platform technology for seed treatment with active ingredients for crop protection enabled by plant viral nanoparticle and traditional cellulosic pulping processes. Establish the scientific foundation with host communities in Eastern Africa insuring translational implementation of the prototype concept for eliminating the biotic stresses in subsistence farming that reduce crop yield and quality.

NCSU will assist with analysis and management of next Generation sequencing data, expression data, and other data as requested by ORNL. Duties may include activities such as analytics, programming, and algorithm development, and staff education. All data sets and documentation generated during data analysis will be retained by ORNL for the duration of the project

Intellectual merit: We propose to fully characterize the role of RNA-RNA and RNA-coat protein (CP) interactions in the Red clover necrotic mosaic virus (RCNMV) origin of assembly (OAS). The RCNMV genome is split between two positive sense single-stranded RNAs. The two genomic RNAs base-pair with each other to serve as a key molecular switch in the virus life cycle. The RNA-2 34-nucleotide stem-loop structure termed the trans-activator (TA) base-pairs with the RNA-1 trans-activator binding site (TABS) forming a stable trans-pseudoknot. So far, subgenomic RNA synthesis, CP expression, RNA-2 replication, virion assembly, and possibly even suppression of gene silencing functions have been assigned to the RNA-2 TA alone or complexed with the RNA-1 TABS. We will fully determine the role of the TA and the TA:TABS interaction in virion content and assembly. RCNMV exists as two virion populations: i) biologically relevant virions containing a copy of RNA-1 and RNA-2 and ii) virions containing multiple copies of RNA-2 whose biological role, if any, is not known. The RNA-2 34 nucleotide TA is necessary and sufficient to direct virion assembly and is therefore deemed to be the OAS. The main question to be answered in this proposal is, does the TA itself possess OAS activity or does it need to be complexed with another RNA? We hypothesize that the RNA-2 TA must base pair with RNA-1 at the TABS to form a functional OAS and direct assembly of infectious virions. We further hypothesize that the RNA-2 TA interacts with other RNA-2s in trans with a TABS-like element to form multiple copy RNA-2 virions. We will also address the packaging arrangement of a second small sgRNA. We will test these hypotheses using an in vivo assembly assay coupled with genetic studies and structural and biochemical approaches. The three specific aims to be accomplished in this study are to: 1. Determine if the OAS consists solely of the RNA-2 TA or the TA complexed to RNA-1; 2. Determine structure of the OAS RNA or RNA-RNA interactions that CP recognizes and initiates assembly at; 3. Identify other RNA-RNA interactions involved in packaging of the other viral RNAs; Broader impacts: A long standing question in virology is how do viruses with multiple genomic segments package the genome into virions? What is discovered in the context of this project will certainly be applicable to other multicomponent icosahedral RNA plant viruses and likely provide insights into similar animal viruses as well. This RCNMV TA/TABS switch structurally and mechanistically parallels both the Simian retrovirus type-1 frameshifting pseudoknot and the Human immunodeficiency virus RNA kissing hairpin complex involved in virion assembly. That RCNMV possesses these features conserved in both form and function with a number of health related RNA viruses in a single element attests to the unique opportunity offered by research on this plant pathogen. The elucidation of this mechanism may lead to the development of molecular control strategies for virus diseases based on the specific disruption of virion assembly. This study will extend the role of RNA-RNA interactions beyond transcriptional activation to virion assembly and further suggest additional important regulatory and structural roles for either cis or trans RNA-RNA interactions. Finally, plant viruses only cause a disease when they form a systemic infection. For most plant viruses, virion formation has been implicated in this process, but not definitively established. The elucidation of the RCNMV OAS will allow for a direct test of the need for virion formation for long distance transport and systemic infection. Education: High school, undergraduate, and graduate students, technicians, post-doctorals and visiting scientists will continue conducting research in the laboratory. The PI is recruiting a pacific-island female to the laboratory to be the funded graduate student on the project. The PI has a robust collaboration with Dr. Smith, UNC Greensboro, an HBCU, and will continue to accept her rotation students through the lab

This proposal describes a summer undergraduate student program at Adam Mickiewicz University (AMU) and the Institute of Bioorganic Chemistry of the Polish National Academy of Sciences (IBCh) in Poznañ, Poland. The program involves an educational and undergraduate research component to research projects that involves collaborative research on problems of general interest for RNA structure and function. The theme of the research is the study of structures of oligonucleotides that are involved in viral coat protein assembly using methods that include modified fluorescent oligonucleotides. The PI and co-PI have recently embarked on program of research that involves the development of novel plant viral nanotechnology. We have collaborated with the Polish research groups of Prof. Bohdan Skalski (AMU) and Prof. Ryszard Adamiak (IBCh) for the past six years. This collaboration has included joint publications, patents, reciprocal visits to laboratories, seminars collaborators universities, short courses at AMU visits by graduate students and a summer undergraduate research project initiated in 2006. Over the course of the past 6 years the PI, co-PI and Polish collaborators have developed the undergraduate summer research program described here. The program will benefit from synergistic projects that have common features. We have found a compromise the benefits the research projects in collaborators laboratories while providing a coherent training for students.