Marce Lorenzen

The long-range goal of our proposed research is to develop novel, sustainable, and biologically-based strategies for controlling thrips and orthotospoviruses in agricultural croplands and greenhouses. The project will address the Pests and Beneficial Species in Agricultural Production Systems goal to ���advance knowledge of invasive or established plant pests and associated beneficial species leading to innovative and biologically-based strategies to manage pests.��� The aim of this proposal is to functionally characterize novel thrips proteins and virus-responsive gene networks associated with thrips vector competence. In other words, we plan to identify biotic factors affecting the ability of thrips to transmit TSWV, which is in line with the priorities of this NIFA program area.

The next generation of plant virus vectors for delivering proteins and RNAs to plants and insects is rapidly developing. In the last five years, there were advancements in technologies that enabled the creation of infectious virus clones for negative strand viruses including the plant rhabdoviruses, tospoviruses, and emaraviruses. These viruses replicate in their plant hosts and insect vectors creating a complex situation for understanding risks associated with using these types of viruses as delivery systems. The plant rhabdoviruses are of particular interest as virus vectors because they can stably carry large cargo. Recent experiments have shown that plant rhabdoviruses can be used to deliver RNA, proteins (e.g., GFP, Cas9), and gRNAs to plants. There is significant interest in using rhabdoviruses for genome editing in recalcitrant plant and insect hosts. We propose to use our recently developed infectious clone for Maize mosaic alphanucleorhabdovirus (MMV) as a model for understanding risks associated with negative-strand RNA viruses. Our research goals will enable us to better understand the ability of GE MMV to replicate in plant and insect hosts thus addressing the need to monitor and understand dispersal of GE viruses (Area 2). We will also use this basic knowledge to attempt to develop viruses with modified ability to infect plants and insects, depending on the host targeted for gene modification (Area 1). We will also use the MMV clone in experiments to determine the potential for genetic exchange between virus clones and wildtype (wt) viruses and characterize the full host range of the virus in grasses and selected planthoppers thus addressing the goals of Area 3.

Thrips species such as Frankliniella occidentalis vector a variety of plant-infecting viruses. The proposed project aims at developing genome-editing tools for thrips, and using these methods to decrease vector competence, thereby reducing reliance on conventional chemical management strategies for vector-transmitted diseases. We hypothesize that editing thrips genes that encode proteins known to interact directly with tomato spotted wilt virus (TSWV) or respond to TSWV infection during acquisition will produce a more ���������������TSWV-resistant������������������ insect, manifested by reduced transmission efficiency. Our research will address the USDA-NIFA challenge of developing more sustainable, productive and economically viable plant production systems, while also addressing each of CALS������������������ five core strategic themes: enhancing production, quality, and profitability of food; ensuring environmental stewardship and sustainability; creating a safer food supply; improving human health and well-being; and preparing students for leadership and success in the global workforce through graduate and undergraduate research opportunities in the fields of agricultural biotechnology and extension.

Marc�� Lorenzen, Ph.D. will be responsible for the overall coordination and supervision of all aspects of the study. Dr. Lorenzen will be responsible for overseeing and/or performing the procedures necessary to achieve the following goals. Specifically, Dr. Lorenzen and/or her team will: 1) collect tissue from late larval stage and mid-adult stage Apis mellifera for transcriptome sequencing; 2) isolate RNA of sufficient quality for Illumina TruSeq library prep; 3) oversee submission to NCSU���s Sequencing Center; 4) obtain and quality check resulting FASTQ files; 5) process sequence data to provide BASF with the following: a) FASTA files representing each sequence library, b) separate and combined transcriptome assemblies, c) fully-annotated gene lists and d) information on relative expression levels. Dr. Lorenzen will also be responsible for any reports or presentations that may be required.

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.

Food security is increasingly threatened by a burgeoning population and environmental factors such as climate change, and alternative food sources are increasingly in demand. One option for alternative food sources is the incorporation of insects into food products, already a standard food for 80% of the world������������������s population, but relatively novel in the western world. NCSU has partnered with the United States Department of Agriculture and All Things Bugs LLC to genetically engineer two of the most commonly farmed insects, the yellow mealworm (Tenebrio molitor) and the house cricket (Acheta domesticus). Our goal is to produce insects strains having increased disease resistance and enhanced protein and nutrient profiles.

Bacillus thuringiensis (Bt)-based pesticides have been deployed as sprays since the 1950������������������s, and as transgenes since 1996. As of 2017, Bt varieties accounted for over 80% of corn and cotton acreage. However, environmental, health, and economic benefits of using this pesticide are threatened by insects that are developing resistance. From 2005-2017, 9 species have developed resistance to Bt. Many alternatives to using Bt are more harmful to non-target animals. Bt is non-toxic to vertebrates like humans and birds, and is even selective in the types of insects targeted, so can be used without harming beneficial insects. However, the molecular mechanisms of Bt toxicity and resistance in beetles are not understood. The overall goal of this research is to use a genome modification method, CRISPR/Cas9, to investigate Bt toxicity and resistance in a model organism, the red flour beetle (Tribolium castaneum). A better understanding of CRISPR/Cas9 in Tribolium will pave the way for genetic research in additional agronomically-important beetles. Data on the molecular mechanism of toxicity will facilitate methods for resistance management. This research addresses plant health and production and plant products, as it will preserve the benefits of Bt foliar sprays and transgenic crops, thereby protecting food resources in the US. Broadly, this research will facilitate the USDA in its mission to solve problems in farm sustainability, including food safety, production, quality-of-life for farmers, and environmental quality. The goals of the EWD Predoc will be supported by enhancing my leadership abilities as a future ag-biotech researcher and regulatory affairs specialist.

There is an inverse relationship between insecticide resistance and customer acceptance of insecticide usage. Specifically, while insecticide resistance continues to increase, public acceptance is decreasing. Through this agreement, our goal is to develop new, low cost, eco-friendly control strategies for coleopteran storage pests based on oral RNA interference (RNAi). Since the process relies on sequence homology, a dsRNA designed to target an insect gene will not trigger an RNAi effect in humans, if properly identified through bioinformatics. In fact, a dsRNA designed to target a gene in the red flour beetle, Tribolium castaneum, will have no effect on a Monarch butterfly. The sequence specificity of RNAi allows us to target a particular pest species, while leaving all others unharmed. NCSU scientists will work with ARS scientists to evaluate the potential of oral RNAi to target specific genes in T. castaneum using bacterial and yeast systems. ARS scientists have identified a number of potential targets in the gut of larvae and adults, the feeding stages of T. castaneum. The team will incorporate dsRNA into biological organisms, such as Escherichia coli and Saccharomyces cerevisiae, and evaluate the efficacy of different delivery vehicles and targets in T. castaneum to develop new products for the control of damaging coleopteran storage pests.

RNA interference (RNAi) is a sequence-specific gene-silencing mechanism that is initiated by the introduction of double-stranded RNA (dsRNA) into a cell. RNAi is a powerful tool for studying gene function but also holds great potential as a mechanism for insect control. RNAi studies in the western corn rootworm identified two essential genes, V-ATPase and Snf7, that when targeted by dsRNA cause death. Here we propose to evaluate the use of RNAi as a control method for the small hive beetle (SHB), Aethina tumida (Murray). The SHB is a pest of honey bee hives, and causes structural damage to the hive, feeds on pollen and honey bee brood, and is a vector of American foulbrood (Paenibacillus larvae). Results from this study will provide a baseline of molecular data (e.g. gene expression) and may lead to an environmentally friendly alternative to pesticide-based control of SHB. The project will also initiate a dialog between state beekeepers and researchers about the potential of using molecular tools to control arthropod pests in apiculture. Moreover, this research addresses each of CALS������������������ five core strategic themes: enhancing production, quality, and profitability of food; ensuring environmental stewardship and sustainability; creating a safer food supply; improving human health and well-being; and preparing students for leadership and success in the global workforce through graduate and undergraduate research opportunities in the fields of agricultural biotechnology and extension.

The proposed research is aimed at revealing the molecular genetic mechanism by which selfish genetic elements increase in frequency within populations although they provide no fitness advantage. The maternal-effect selfish elements under investigation are described as encoding two linked components, a maternal poison and a zygotic antidote. While a number of maternal-effect selfish elements have been described in beetles and mice, the molecular genetic mechanism by which such elements function remains a mystery. Such elements are of considerable interest not only from the viewpoint of basic evolutionary and population biology, but also for their potential application as a genetic drivers, for example in the genetic control of vector-borne diseases. Therefore we propose to utilize high-resolution recombinational mapping to positionally clone a selfish-genetic element from the red flour beetle and through comparison with a previously cloned element elucidate the molecular mechanism that underlies their selfish behavior. We plan to apply this knowledge to the improvement of gene drive systems for use in genetic pest management.