Tim Sit

Smallholder farming practices in sub-Saharan Africa (SSA) include land-raised seed (piece) use, continuous cultivation (often monoculture) with limited inputs, and virtually non-existent seed (piece) treatment techniques. Yam (Dioscorea spp.) is the primary example of this cropping system and is an extremely important and valuable crop for smallholder farmers in SSA. These practices result in nutrient-depleted soils, nematode infestation, and ultimately low crop yields. Reducing the nematode population in crop soil will dramatically increase crop yield and quality. In many cases, smallholder farmers in SSA lose greater than 50% of their crop to plant-parasitic nematodes, primarily due to lack of available and affordable control options. For this BMG GCE Phase III proposal, we will build upon our promising results from Phase II trials, including increased yields and higher tuber quality and storability for both yam and potato, and use our expertise and connections for pulping banana fiber to validate and prepare for commercialization a developing-world transferable product platform enabling a field deployable paper-like seed (piece) treatment to combat plant pathogenic nematodes. In addition, this platform will be amenable to delivering other crop production moieties, including natural products and oils, necessary minerals and nutrients, or insecticides and fungicides, with its application. Our laboratories������������������ expertise in nematology and lignocellulosic fibrous materials enables us to target the delivery of beneficial small molecules during seed (piece) germination and plant establishment. Importantly the incorporation of active ingredients into a lignocellulose matrix, such as banana tissue paper, allows for widespread distribution of crop protection agents without interfering in smallholder farming practices. The shelf-stable light-weight banana tissue paper can be applied at the point of seed (piece) planting where farmers can use the concept of ����������������wrap and plant��������������� with their own seeds/pieces. Our ���������������wrap and plant������������������ product will be an ����������������active��������������� paper sheet pretreated with ultralow concentrations of active nematicidal ingredient to simply wrap and protect the seed (piece) at planting. The localization of active ingredients carried directly within the paper targets specific plant pathogenic nematodes versus beneficial organisms. Nematodes are primarily a seedling disease, so protection early is critical to the success of the crop, although post harvest losses do occur in yam due to the yam nematode (Scutellonema bradys). Reduction in nematode populations by deploying the ���������������wrap and plant������������������ product protects yam from significant infections that may lead to these post-harvest losses. Our ultimate goal is to validate the ���������������wrap and plant������������������ product in Phase III and to translate the product manufacture to a regional African company for commercialization and distribution.

Our goal is to develop rapid and transient approaches to modify traits in growing maize crops using engineered viruses introduced by insect transmission for gene expression, silencing and editing. Improvement of maize using current strategies requires several years and the state of the art modifiable virus for maize is based on brome mosaic virus, which is unfeasible to contain due to its ready mechanical transmission to a broad range of hosts. Key technical challenges are: 1) identifying and developing virus systems that allow stable expression of large heterologous sequences; 2) developing specific, efficient, and controlled insect delivery; 3) limiting spread of modified viruses; and 4) modifying phenotypes in maize at relevant developmental stages. We will address these by testing multiple virus-insect systems and utilizing expertise in rice CRISPR/Cas9 and insect transgenic development. Planned research spans 4 years of work by a team of 11 experts, costing a total of $9.68 million. Successful completion will identify improved tools to elucidate plant-virus-insect gene functions and molecular interactions, flexible genomics tools for silencing, expression and editing, and ultimately allow real-time rapid response to biotic and abiotic stresses in the field and reduce input and breeding costs for maize.

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.

The long time period from seed to seed for tobacco is a chronic limitation for classical breeding programs in their quest for introducing desirable traits. The Arabidopsis thaliana FLOWERING LOCUS T (FT) gene product was shown to be responsible for induction of flowering. Both the FT protein as well as its mRNA are capable of long distance transport to the site of action. Flowering times were dramatically reduced when tobacco plants were stably transformed with an Arabidopsis FT gene, decreasing flowering time from an average of 87-138 days for non-transgenic plants to ~39 days for FT expressing plants. Virus-based FT expression offers several advantages over stable transformation-based strategies, as this allows the flexibility of inducing early flowering for any plant, not just ones that have been previously transformed with FT or crossed with a plant carrying the FT transgene. Furthermore, transient expression eliminates the need to segregate away the transgene once the early flowering phenotype is no longer desired. The use of viral vectors as a means for expressing FT in tobacco, however, has yielded inconsistent results and has yet to be proven as a reliable means to induce early flowering. This may have been due to the non-optimal host range of the various viral vectors available. In addition, given the inherent mobility of the FT protein, the actual need for a viral vector is questionable, and an effective early flowering induction system may be possible merely through Agro-infiltration of the leaves of intact young plants. However, due to potential aversion to using Agrobacterium based expression systems for breeding purposes, we propose it would be advantageous to attempt to develop two systems, one based on transient expression via Agro-infiltration and the other using an RNA-based viral vector specifically adapted to tobacco.

Plant parasitic nematodes are one of the world's major agricultural pests, causing in excess of $125 billion in worldwide crop damage annually. Nematodes are primarily a seedling disease, so protection early is critical to the success of the crop. Traditionally, control has depended on highly toxic contact and fumigant pesticides which have now been restricted or eliminated in the United States by the EPA. Issues such as ground water contamination, mammalian and avian toxicity, and residues in food have caused much tighter restrictions on the use of these agricultural chemicals. Current corporate focus for nematode control in major crop plants (maize, soybean, cotton, etc.) is largely seed treatment approaches that provide early protection, which is critical for nematode management. In theory, seed treatment for nematode control is an excellent idea, but in practice it has been largely ineffectual due to delivery problems post-germination. We propose to solve this problem by concentrating nematicides within plant virus-based nanoparticles (PVN) which would be incorporated into a biodegradable cellulosic matrix as a seed coating. The matrix would dissolve and spread with the growing root bundle providing a zone of protection not available with current seed coatings. Furthermore, this approach enables a reduction in the amount of active ingredient applied, exposes the target organism to a much higher concentration of active ingredient, and substantially reduces non-target effects and environmental impact. This proposal is a significant advancement of a project currently funded by the Bill & Melinda Gates Foundation Grand Challenges Explorations Round 8 (Field-Deployable Nutrient-Rich Matrix for Crop Protection). It differs from the current project by testing a larger range of nematicides and crop species specific for the United States (vs. Africa), focusing on first world cropping systems, while also seeking to determine the fate of the PVN within the environment. The overall goal of the project is the development and characterization of PVNs to enhance the performance of a range of nematicides while reducing their environmental impact through decreases in application levels. Specifically, we will: 1) Determine the ability of PVNs to cargo nematicides. We will test a broad range of therapeutically approved nematicides (including ones not currently used for agricultural purposes) for their ability to be loaded onto and released from PVNs. Bioassays using formulated PVNs will be performed on the model nematodes Caenorhabditis elegans and Meloidogyne hapla. Prior to application in soil tests, deactivation procedures for the viral genome will be assayed for their effect(s) on viral infectivity, PVN integrity and nematicide loading and release. 2) Characterize changes in nematicide properties within loaded PVNs in soil. The concentration of nematicides within the PVN will undoubtedly alter their properties with respect to solubility, mobility, persistence and minimum effective dosage. These differences will be assayed by comparing free nematicide to PVN bound nematicide in various soil types via flowthrough assays. Bioassays will then be performed on the collected fractions. 3) Develop and optimize cellulose matrices of various compositions for PVN uptake/release. By controlling packing density via fiber diameter and PVN concentration, locality of the PVN distribution throughout the fiber, biodegradability of the matrix, and functionalization protocol, we will understand the enabling features of the PVN coupled with its carrier matrix. Ultimately, this project will produce a seed treatment that is both efficacious and economical that would both revolutionize and enhance the nematicide market while achieving a crucial movement toward environmentally sustainable agriculture.

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.

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