Dorith Rotenberg

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.

We propose to develop new methods for tracking the spread of plant pathogens through agricultural landscapes using population genetic data. Because plant pathogens spread across complex landscapes, our approach will build on network models from spatial epidemiology that provide the flexibility needed to track epidemic dynamics across multiple scales and locations. Network models will be combined with phylogenetic approaches for estimating spatial spread based on the genetic relatedness of pathogens sampled at different geographic locations. These methods will then be implemented in high-performance, user-friendly software for analysis and web-based visualization. We aplan to apply our approach to study the spatial epidemic dynamics to three crop pathogens of major economic importance: Barley yellow dwarf virus, the aflatoxin-producing mold Aspergillus flavus and the downy mildew Pseudoperonospora cubensis. By synthesizing advances in spatial epidemiology and population genetics, our approach will provide next-generation software tools that will help reveal the dominant pathways by which these pathogens spread and identify major geographic sources that future control strategies can target.

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.

Host preference and performance of arthropod pests on plants, i. e., foraging, feeding, and oviposition, are tightly linked to host quality (primary plant chemistries) and herbivore-inducible host defense mechanisms (secondary plant chemistries) that serve to limit arthropod performance on plants. In turn, herbivorous arthropod populations have evolved mechanisms to counter these host defense chemistries. At the center of these counter-defense mechanisms are diverse and species-specific repertoires of salivary gland (SG) proteins that are secreted during salivation and feeding that can modulate or dampen host defenses in such a way to significantly enhance herbivore colonization (termed ����������������effector��������������� proteins). The repertoire of SG-enriched proteins is virtually unknown for one of the most important crop pests and plant-virus vectors, the western flower thrips (WFT) (Frankliniella occidentalis). Furthermore, the plant-pathogenic tospovirus, Tomato spotted wilt virus (TSWV), is transmitted by WFT in a persistent propagative manner, whereby the virus replicates in the salivary glands prior to inoculation into the plant during thrips feeding. Two questions drive our research proposal: 1) Does TSWV infection of thrips SGs modify the abundance or composition of the repertoire of SG-enriched proteins in thrips, and 2) do TSWV-responsive SG proteins modulate the ability of the plant to respond to herbivory or the insect to perform and reproduce on the plant host? The goal of our integrated and collaborative research project is to identify and quantify the direct effect of TSWV infection on thrips SG-gene expression at the transcriptome (Aim 1) and proteome (Aim 2) levels and to determine to what extent the suite of TSWV responsive genes modulate the molecular and biological interactions between the insect vector and the plant host (Aim 3).

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.

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 was to develop effective multigenic, broad-spectrum resistance to 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 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. If the plants are resistant to RB-TSWV, in future years we will transfer the hairpins into popular California tomato varieties by breeding or additional transformation events.

Distinct steps in virus transmission by insect vectors provide targets for disease cycle disruption. A specific viral protein(s) is required for attachment and/or entry into the insect. Exploiting viral attachment proteins (VAPs) is a logical approach for reducing transmission. Tomato spotted wilt virus (TSWV) and related tospoviruses are a threat to U.S. agriculture and food security. TSWV is transmitted in a persistent propagative manner by thrips and acquisition is mediated by the interaction between the virus glycoprotein GN, which serves as a VAP, and the thrips midgut. We demonstrated that a soluble form of the GN protein (GN-S) binds to thrips midguts and inhibits virus acquisition and transmission. Additionally, transgenic tomato expressing GN-S significantly lowered virus titers in larval and ensuing adult thrips, and reduced transmission efficiency. The central aim of this proposal is to characterize the interaction between thrips and GN-S for understanding the biology and potential deployment of control strategies aimed at disrupting virus transmission. The specific objectives of this proposal are to: 1. Determine the mechanism of transgenically-expressed GN-S inhibition of TSWV acquisition and transmission by thrips; 2. Test GN-S transgenic tomato plants for the ability to inhibit transmission of related and newly emerging tospoviruses; and 3. Define the domains of GN required for binding to thrips midguts. Two research focus areas of the Plant-Associated Microorganisms priority area will be addressed: i) elucidation of molecular mechanisms used by microorganisms to interact with plant hosts; and ii) examining epidemiological factors that influence disease spread.