Anna Whitfield

Previous work has demonstrated that a protein on the surface of tomato spotted wilt virus binds to a thrips gut receptor to facilitate virus entry and replication. It is also known that the specificity of Bt toxin depends on targeting Bt to an insect gut receptor by a specific protein. We are using standard molecular techniques to produce Bt that contains the virus surface protein in place of its native targeting protein. The rationale is that the Bt fusion will bind to thrips guts (and only thrips guts because the virus is not transmitted by any other insect) and create a specific (no harm to beneficial insects), benign (years of use of Bt have shown it to be safe) and effective pesticide. A robust transmission assay will be used to experimentally evaluate Bt fusions for control of thrips and orthotospoviruses. A thrips-specific Bt was recently expressed in cotton and this may offer a second approach for thrips control in ornamentals. The objective of this project, therefore, is to test various constructs for effects on blocking transmission and acquisition of orthotospoviruses in host plants.

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.

Emerging plant disease and pest outbreaks reduce food security, national security, human health, and the environment, with serious economic implications for North Carolina growers. These outbreaks may accelerate in coming decades due to shifts in the geographic distributions of pests, pathogens and vectors in response to climate change and commerce. Data-driven agbioscience tools can help growers solve pest and disease problems in the field more quickly but there is an urgent need to harness game-changing technologies. Computing devices are now embedded in our personal lives with sensors, wireless technology, and connectivity in the ����������������Internet of Things��������������� (IoT) but these technologies have yet to be scaled to agriculture. Our interdisciplinary team will build transformative sensor technology to identify plant pathogens, link local pathogen data and weather data, bioinformatics tools (pathogen genotypes), and use data driven analytics to map outbreaks, estimate pest and pathogen risk and economic damage, in order to coordinate response to emerging diseases, and contain threats. Sensor-supported early and accurate detection of pathogens before an outbreak becomes wide-spread in growing crops will significantly reduce pesticide use and increase crop yields.

Tomato spotted wilt virus (TSWV) is a globally-distributed virus that is estimated to cause annual crop losses of more than $1 billion. Like other members of the order Bunyavirales, TSWV (family: orthotospoviridae) is an enveloped single-stranded negative-sense RNA virus with a tripartite genome. The medium (M) segment encodes the virus envelope glycoproteins, GN and GC, that are required for insect transmission. TSWV is transmitted by the insect vector thrips, and the most widespread and efficient vector is Frankliniella occidentalis, the Western flower thrips (WFT). The unique biology of the TSWV-thrips interaction, allows a narrow window of opportunity for virus acquisition during larval development, and this presents a unique target for disrupting the virus transmission cycle. The TSWV glycoprotein, GN, is involved in virus binding to the gut and represents a target for the development of new anti-viral and insecticidal molecules to disrupt the transmission process. At this time, the critical knowledge gap at the molecular level of TSWV-thrips interactions and the structural biology of the virus preclude the rationalization of efficient solutions for this acute agricultural problem. Here we propose a synergistic and multidisciplinary research plan to decipher the structural biology of GN and molecular mechanisms underpinning TSWV-thrips recognition. The specific research aims are to: 1) Determine the molecular structure of TSWV GN and study its oligomeric states and their equilibrium in solution, 2) Identify WFT proteins that interact directly with TSWV GN and determine the GN molecular landscape required for the interactions with these proteins, and 3) Design, develop and functionally test peptide inhibitors for the prevention of TSWV acquisition in WFT. Our preliminary results include an initial model for TSWV GN structure, the first near-atomic resolution structure for an enveloped plant virus. Our screen for thrips proteins that interact with GN has identified a promising candidate interactor that was validated using multiple approaches. This research plan brings together the expertise of each PI (structural biology of viral glycoproteins (MD) and molecular TSWV-WFT interactions (AEW)), to fully interrogate the structure-function interplay of TSWV GN protein will reveal the molecular mechanism and the physicochemical requirements for virus assembly and its recognition by the thrips. This proposal offers unprecedented insight into the TSWV-thrips biology and it will be an engine for developing transmission-disrupting molecules for control of tospoviruses and thrips and integrate them into multi-faceted disease management programs.

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.

Tomato spotted wilt (TSW), spread by thrips, is a widespread and destructive disease worldwide. It is controlled in commercial production by the use of hybrids with the Sw-5b resistance gene. However, the resistant-breaking (RB) strains of the virus not controlled by the Sw-5b gene have become prevalent in some regions including processing tomato production areas in California. In order to mitigate the damage caused by RB strains of the tomato spotted wilt virus (TSWV), a polygenic host resistant incorporated into processing and fresh market tomatoes seems critical. In this research, we aim to (1) utilize DNA marker-assisted breeding to stack the Sw-5b gene with other TSWV resistance genes, Sw-1 and Sw-7, (2) confirm the resistance in a greenhouse with different RB strains of the virus, and (3) evaluate the resistance and horticultural performance in grower���s field. We will utilize conventional breeding approaches expedited with marker-assisted selection to develop new tomato lines that can be used by public and private processing tomato breeding programs. Resistance will be confirmed in grower fields with high TSW incidence. This project will provide important tools to prevent TSW and increase production and profitability by growing improved conventional cultivars with superior fruit quality and multiple disease resistances.

Maize mosaic virus can be used to deliver protein, Cas nucleases, and sgRNAs for high level expression in maize. We have developed constructs that will deliver sgRNA and Cas9 to tobacco and maize, and are optimizing the carrying capacity and delivery strategies. This ground-breaking technology for genome editing in maize can facilitate experiments to improve crop resistance to biotic and abiotic stressors. MMV is also a useful platform for delivery of proteins (insecticidal proteins, etc). This project aims to optimize the MMV delivery system by optimizing reporter gene placement in the virus genome. We will also test the sgRNA/Cas9 construct in maize plants for success in genome editing.

Tomato spotted wilt virus (TSWV) and related thrips-borne orthotospoviruses are some of the biggest threats to production of food and ornamental crops, and new orthotospovirus-caused diseases are emerging in the U.S. and globally. Orthotospoviruses have the capacity for rapid genetic change due to their segmented genome that allows for reassortment and mutations caused by their error-prone polymerase. Genetic resistance is one of the most effective control strategies for managing orthotospoviruses, but there are multiple examples of resistance gene breakdown. The goal of this project is to develop effective multigenic, broad-spectrum resistance to TSWV, resistance-breaking TSWV, and other orthotospoviruses. Bioinformatic analysis of virus sequences in GenBank enabled us to identify the most conserved sequences for each open reading frame (ORF) of the TSWV genome. Comparison to other orthotospovirus species revealed that those sequences were often conserved within virus clades and some had well-documented biological functions. We constructed 5 hairpin constructs, each of which incorporated sequences matching portions of all 5 ORFs. Tomato plants expressing the hairpin transgene were challenged with TSWV by thrips and leaf-rub inoculation and three constructs provided strong protection against TSWV and tomato chlorotic spot virus (TCSV). Because these hairpin constructs target all five viral open reading frames (ORFs), we expect them to confer protection against newly-emerged resistance-breaking (RB) isolates of TSWV. We will test these promising transgenic tomatoes against RB-TSWV using repeated mechanical inoculation and thrips transmission. We will analyze the virus populations to determine how the virus responds to this new selection pressure. Hairpin constructs are an effective way to protect plants from multiple orthotospoviruses and the targeting of all five viral ORFs in each single construct is expected to increase the durability of resistance. We will also generate third generation plants (T3) for additional testing of virus resistance and transgene stability. If the plants are resistant to RB-TSWV, in future years we will transfer the hairpins into popular California tomato varieties by creating new transformants and/or through conventional breeding. This project will provide new sources of TSWV resistance for integration into breeding programs that can be combined with other traditional resistance genes to provide multigenic protection from emerging viruses.

Our goal is to develop new and effective control strategies for thrips and tospoviruses. To achieve this goal, we are exploiting the specificity of the thrips-tospovirus interaction. Previously, we found that the Tomato spotted wilt virus (TSWV) Gn protein binds directly to thrips guts and blocks virus transmission. We are testing fusion proteins of Bacillus thuringensis (Bt) toxins and Gn for activity against thrips and defining the Gn binding domains to develop better toxin constructs. Other strategies for control focus on using dsRNA molecules to for insecticidal activity against thrips. We are also testing transgenic antiviral constructs for activity against tospoviruses of importance to the floriculture industry such as Impatiens necrotic spot virus (INSV).