INTEGRATIVE APPROACHES TO UNDERSTANDING HOW INSECT VECTORS AND PLANT VIRUSES INTERACT AND MANIPULATE THEIR PLANT HOSTS

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: COMPLETE
• Funding Source: AFRI COMPETITIVE GRANT
• Reporting Frequency: Annual
• Accession No.: 1016609
• Grant No.: 2018-67013-28495
• Cumulative Award Amt.: $1,060,000.00
• Proposal No.: 2018-05351
• Project Start Date: Sep 1, 2018
• Project End Date: Aug 31, 2023
• Grant Year: 2018
• Program Code: [A1171]- Plant Biotic Interactions

Recipient Organization
UNIVERSITY OF CALIFORNIA, DAVIS
410 MRAK HALL
DAVIS,CA 95616-8671

Performing Department
Entomology/Nematology

Non Technical Summary
This project supports the mission of the Agricultural Experiment Station by addressing the Hatch Act area(s) of: plant and animal production, protection, and health; sustainable agriculture; molecular biology; and, biotechnology. Host preference and performance of pest insects on plants are linked to host quality (primary plant chemistries) and inducible host defenses (secondary plant chemistries). In turn, plant-feeding insects have evolved mechanisms to counter host defense chemistries. At the center of this interaction are diverse and species-specific repertoires of salivary gland (SG) proteins secreted by insects during tissue probing or feeding that can induce or suppress host defenses, often enhancing insect fitness and profiting pathogens they transmit ("effectors"). Almost nothing is known about SG-enriched proteins for the Western flower thrips (WFT), an insect that damages plants by feeding and transmitting the plant virus, Tomato spotted wilt virus (TSWV). This dearth of information restricts advances in developing host plants with WFT and TSWV resistance and threatens sustainability of existing resistant plant lines. This proposal describes an integrated investigation of how TSWV infection affects expression of SG-enriched genes on transcriptome and proteome levels, and determines the extent to which TSWV responsive genes modulate molecular and biological interactions between WFT, TSWV and plant defenses. Aim 1: Test how TSWV infection status, developmental stage and sex effect expression of WFT SG-enriched genes. Previous whole body transcriptomes show developmental stage and infection status affect gene expression. Our SG-specific investigations reveal a decrease in expression of one SG-enriched gene in infected adult females. These discoveries will be extended through comprehensive transcriptomic studies of SG from non-infected and TSWV-infected WFT larvae and adults (male and female). Aim 2: Test how TSWV-infection status, developmental stage and sex affect the proteome of WFT salivary gland tissue and saliva. This analysis will reveal the composition of proteins, including SG-enriched proteins with known involvement in feeding (i.e. extra oral digestion, effector activity) and novel WFT-specific proteins with no known function. Aim 3: Test how differentially expressed proteins from non-infected and infected WFT may vary in modulating plant defenses against the insect and the virus it transmits (TSWV) by phenotypic-screening of plants transiently-expressing SG-enriched proteins. Aims 1 and 2 will guide selection of candidate genes for phenotypic analysis. Specific hypotheses will be tested in Aim 3 to discover plant responses to expression of SG-enriched proteins and how these responses, as well as TSWV-infection status modulate WFT fitness, physiology and virus transmission. Host preference and performance of pest on plants are linked to host quality and inducible host defenses. In turn, plant-feeding insects have evolved mechanisms to counter host defense chemistries. At the center of this interaction are diverse and species-specific repertoires of salivary gland proteins secreted by insects during probing or feeding that can induce or suppress host defenses, often enhancing insect fitness and profiting pathogens they transmit ("effectors"). Although critical for creation of durable host plant resistance against WFT and TSWV, we know little about WFT's salivary protein repertoire. Nor do we have fundamental information about interactions between SG-enriched transcripts, salivary proteins and TSWV in the SGs; or the potential role of thrips salivary proteins as effectors, eliciting and/or suppressing plant immune systems against the insect and TSWV. There is an urgent need for the research we plan, which will discover how TSWV infection modulates SG-enriched transcripts on transcriptome and proteome levels, and determine the extent to which TSWV responsive genes alter molecular and biological interactions between WFT, TSWV and plant defenses. These findings will identify specific targets aimed at interfering with WFT feeding behavior and fitness, leading to TSWV acquisition, inoculation and infection of plants.

Animal Health Component

20%

Research Effort Categories

Basic

80%

Applied

20%

Developmental

(N/A)

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
211	1460	1130	20%
211	3110	1130	15%
211	4030	1130	15%
212	1460	1130	20%
212	3110	1130	15%
212	4030	1130	15%

Knowledge Area
212 - Pathogens and Nematodes Affecting Plants; 211 - Insects, Mites, and Other Arthropods Affecting Plants;

Subject Of Investigation
1460 - Tomato; 3110 - Insects; 4030 - Viruses;

Field Of Science
1130 - Entomology and acarology;

Keywords

insect

vectors

and

plant

viruses

Goals / Objectives
Our goal is to explore how fundamental biological interactions between the Western flower thrips (WFT), Frankliniella occidentalis, and a plant virus it transmits, Tomato spotted wilt virus (TSWV) (genus Tospovirus) may modulate insect-plant interactions and host plant defenses of agriculturally significant plant hosts (i.e., tomato). We propose multifaceted, integrated, experiments to address questions about how TSWV infection of WFT affects expression of transcripts enriched in the SGs (SG-enriched transcripts), and the SG proteome and secretome (proteins secreted into saliva). We will tie these fundamental findings to a whole-organism view by asking questions about the impact of differential expression of SG-enriched proteins on plant defenses and WFT physiology, behavior and fitness, thus identifying salivary proteins that may serve to facilitate feeding or as effectors modulating plant defenses. Our previous discoveries provide a strong foundation for this research. First, in whole body WFT developmental stage-specific transcriptomes, TSWV infection perturbed expression of genes associated with WFT processes and functions (37). Second, comparison of the draft WFT genome whole body and SG transcriptomes revealed a subset of SG-enriched transcripts. Third, among these transcripts, sequences encoding signal peptides that are hallmarks of secreted proteins, are present, providing a preliminary list of potentially secreted salivary proteins; and, finally, with qRT-PCR, we detected differential expression of a putative pancreatic lipase gene in TSWV infected WFT SGs that is a key gene of interest.In view of our preliminary findings, we propose to: 1) test the hypothesis that WFT SG-enriched transcripts are differentially expressed in response to development, sex, and TSWV infection. We will achieve this aim through comparative transcriptomics of dissected SGs from non-infected (NV) and TSWV-infected (V) larvae, first instar (L1) and adult WFT (males and females). We specifically chose this approach because next generation sequencing will enable comprehensive characterization of TSWV's effect on expression of SG-enriched transcripts and set the stage for proteomic investigations. The next major component of the proposed research will be to: 2) test the hypothesis that the WFT salivary protein repertoire will vary with development and sex, and TSWV infection may modulate SG-enriched proteins, as well as proteins secreted into the saliva. We will achieve this aim through comparative proteomics of SGs and saliva from NV- and V-WFT. As planned for the transcriptomic investigations, proteomics of dissected SGs will compare NV- and V-larval and adult WFT (both sexes). Secretome proteomics will focus on adult NV- and V-WFT, because we are confident we can collect adequate quantities of saliva for analysis and adults are most important in virus spread. We chose this approach because it provides a holistic view of the salivary protein repertoire, teases out the proteinaceous components secreted in saliva, and reveals effects of TSWV-infection. Finally, guided by results from these aims and our preliminary data, we will: 3) test how differentially expressed proteins from NV- and V-WFT may vary in modulating plant defenses against the insect and TSWV by phenotypic-screening of plants transiently expressing SG-enriched proteins. We will achieve this aim by measuring: expression of markers of plant defense pathways, visible signs of plant defense (i.e., local lesions, yellowing), and WFT responses i.e., fitness components (oviposition, longevity), physiological aspects, feeding, and virus inoculation.This research is exciting because it fills a major gap in understanding how thrips interact with plants and serve as TSWV vectors. Investigations of salivary protein repertoires and potential effectors produced by herbivorous piercing-sucking arthropods have focused on phloem feeders (primarily aphids). Only two investigations, focused on mites, have characterized the salivary protein repertoire of a piercing-sucking arthropod that feeds in mesophyll (9, 10). Our research will deliver insights into the salivary protein repertoire of the WFT, a piercing-sucking insect pest that feeds primarily in mesophyll cells and transmits plant viruses in a circulative-propagative manner. This largely unexplored territory is critical to developing sustainable host plant resistance. Our results will apply directly to agroecosystems because we are working with well-characterized field isolates of WFT and TSWV, and commercial tomato cultivars. We will deliver new and critically needed insights into mechanisms surrounding plant defenses against insect vectors and viruses and strategies to better deploy and protect these defenses for durable resistance.

Project Methods
Thrips rearing and attainment of virus-infected and non-infected developmental staged cohorts. Ullman and Rotenberg maintain WFT colonies originating from an isolate originally collected in Hawaii (1985). Insects are maintained on green bean pods (Phaseolus vulgaris) as described previously. We will standardize WFT rearing conditions, insect feeding and circadian status for transcriptomic and proteomic studies as follows: 1) brush green bean pods on which adult females already laid eggs to remove any newly hatched larvae; 2) place these in a controlled environment chamber at 25°C (16:8 L:D) to generate WFT synchronized for particular developmental stages. To attain NV- and V-WFT, we will use methods described previously. To create V-WFT, we will place half the 0-12-h-old L1s onto bouquets of TSWV-infected Emilia leaves for virus acquisition. Remaining L1s will be placed on non-infected Emilia leaves to attain NV-WFT. After 12-h we will remove Emilia leaves from each treatment and provide L1s with fresh green bean pods. Fresh green bean pods will be provided every 48 hours throughout WFT development to adulthood. Because dissections must be from synchronized, age-specific WFT, we will start a new cohort of V- and NV-WFT every day until dissections are completed. To confirm treatment infection status (V or NV), we will use qRT-PCR to test for virus in subsets of insects at each developmental stage and Tomato spotted wilt orthotospoviurs (TSWV) inoculation using leaf discs as previously described.Thrips SG dissections. Ullman and Rotenberg labs will dissect thrips SGs from WFT for transcriptomic and proteomic studies at a standard time of day for each developmental stage, using actively feeding insects (confirmed by observing green plant material in the midgut). We need 500 ng/sample for RNAseq with coverage of 80 million reads (per LC Sciences). Thus, we estimate needing 50 SGs/sample, which will require 5 hours of dissection. Larval SGs are slightly smaller than adult SGs; hence, we expect to adjust quantities of larval SGs to attain adequate total RNA (70 SGs). Based on calculations derived from proteomic studies of whole thrips, we estimate that SG proteomics will require dissection of 350 L1 SGs, with adjustments for L2s and adults, to attain the five µg of total protein needed.Collecting WFT saliva. Saliva collection for proteomic investigations will be limited to male and female NV- and V-WFT adults. We will sterilize all components of our feeding system and materials and insects will be handled under aseptic conditions. We will create a feeding apparatus by placing 50 thrips inside a 20 ml scintillation vial on which we stretch a layer of Parafilm® (slightly indented to create a pouch). Sterile diet (100 µl) is placed on the stretched Parafilm® and covered with a second layer of stretched Parafilm®. The feeding apparatus will be held in a laminar flow hood. After 24 hours of WFT feeding (confirmed by blue colorant in their mid/hindguts), we will collect the diet and place samples at -80°C until an adequate sample is collected for proteomic analysis.Experimental design for transcriptomic and proteomic investigations. Our transcriptomic and proteomic experiments will complement one another. Transcriptomic and proteomic experiments with SG tissue will be conducted using a factorial design that includes two biological conditions [+/- virus infection], two developmental stages (L1, adult males and females) and three biological replications of the experiment. Proteomic discovery experiments with saliva will be modified by examining only one developmental stage (adults).AIM 1: Test the hypothesis that abundance of WFT SG-enriched transcripts vary with development, sex, and TSWV infection status. Proposed experiments will use RNAseq to comprehensively reveal how WFT development and TSWV-infection status modulates expression of SG-enriched transcripts.Data analysis. Our RNAseq data analysis will identify specific genes and gene networks with differential transcript levels due to TSWV infection in WFT SGs. Prior to contrasting transcriptional profiles, we will examine data quality by FastQC and remove ambiguous or low quality reads by Trimmomatic or CLC genomics (CLC Bio). RNAseq reads will be mapped to the WFT genome using Bowtie and Tophat. Differential gene expression will be determined with the R bioconductor package featuring DESeq based on read-count data and use of CLC Genomics (Qiagen). When needed, we will conduct transformation and normalization. Statistical significance will be based upon Benjamini-Hochberg multiple test correction at 0.05. Gene ontology (GO) terms will be assigned to increased and decreased genes/contigs via Blast2GO and enrichment calculated with a Fisher test. Specific enriched groups of functionally related genes will be determined by the DAVID Functional Annotation system. We will perform analyses using KEGG- and R-spider programs along with BiNGO and TopGo to determine interacting gene networks. We will validate specific genes with qRT-PCR. We used similar methods in previous RNAseq analyses on multiple insect systems, including thrips.AIM 2: Test the hypothesis that composition and abundance of SG-enriched proteins vary with development and sex, and that TSWV infection modulates expression of these proteins. We plan to pursue a quantitative discovery-proteomics approach to catalogue and quantify the repertoire of proteins enriched in SG tissue (tubular and PSG) and saliva of NV- and V-WFT. The proteomics approach will complement the transcriptomics approach (Aim 1) of identifying differentially expressed SG-enriched transcripts in response to virus activity in the SGs and will refine the candidate list for effector-phenotypic screens and physiological studies (Aim 3). We will use virus acquisition methods and infection status confirmation as described in 6.1.1 prior to PSG dissection or saliva collection.Proteomics workflow. The Duke Center for Genomic and Computational Biology - Proteomics and Metabolomics Shared Resource (Durham, NC) will perform the fee-for-service common workflow of Quantitative 1-dimensional ultra-performance liquid chromatography-tandem mass spectrophotometry (1D UPLC-MS/MS) starting with our collected pools of SGs or collected saliva secretions and assist with data analysis.AIM 3: We will use phenotypic screening to test the hypothesis that WFT salivary proteins from NV- and V-insects may differentially modulate plant defenses against WFT and TSWV. We will select candidate genes for testing as WFT effectors using medium throughput effector-phenotypic screening. We will look for peptide signals predicting secretion; however, we are aware that proteins lacking these signal peptides may also be present in saliva and will include such candidates if appropriate. We will initiate our phenotypic screening with candidates we have already cloned. As additional candidates are identified (Aims 1 and 2), we will enter them into our phenotypic-screening pipeline. We expect to include up to 5-10 additional candidates. Our phenotypic screening will examine plant and WFT responses to transient expression of effector candidates.

Progress 09/01/18 to 08/31/23

Outputs
Target Audience:Ullman: Scientific Community ?Unveiling of the Secret Life of Vineyards. Invited comments at the Matthiasson Vineyard, Napa, CA, August 2023. Rotenberg: Invited talks Towards defining molecular determinants of thrips vector competence to transmit tomato spotted wilt virus. Department of Entomology and Plant Pathology, Seminar Series, Auburn University, AL April 17, 2023 ?Towards defining molecular determinants of thrips vector competence to transmit tomato spotted wilt virus. Department of Entomology, Seminar Series, Texas A&M University, College Station, TX. November 3, 2022. Changes/Problems:University of California Davis:Dwindling funds and lack of personnel made further progress difficult during this reporting period. We have worked on completion of two papers and continued to work with Rotenberg and Benoit to complete analyses and develop a paper on the thrips salivary gland transcriptome. University of Cincinnati: None. We have completed two papers and will be assisting in the development of a RNA-seq paper on the salivary glands. North Carolina State University:None. What opportunities for training and professional development has the project provided?University of California Davis No FTE during time. Sulley Ben-Mahmoud is a research scientist at Washington State University and Ryan Packer graduated and worked in the laboratory of Pam Ronald, UC Davis and is now doing his Ph.D. at North Carolina State University. University of Cincinnati No FTE during time. Samuel Bailey has completed his Master's Degree and has obtained employment as a research scientist at a pest control company. North Carolina State University?: Our NCSU undergraduate hourly, Joshua Yueh, Genetics (GN) major, Biotech minor (graduated Spring 2023) enrolled in GN496 (Undergraduate Research Experience) in the Rotenberg lab, and was mentored by Kirsten Lahre, her Research Assistant. He worked on cloning one of his favorite thrips saliva proteins and agro-launching the construct in N. benthamiana. His molecular training in the lab provided the skills and experiential learning to apply for and gain employment in the Department of Microbiology, Icahn School of Medicine at Mount Sinai. How have the results been disseminated to communities of interest?Three papers have been published disseminating specific results to the scientific community. Results have also been disseminated via presentations at various levels. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? A salivary gland transcriptome for second instar larvae and adult males and females, infected and noninfected with TSWV was completed and analyzed. In addition, a salivary gland proteome and secretome was completed and analyzed for thrips adults (male and female), infected and non-infected. The transcriptomic studies reveal sex biased differences between males and females and that the larval transcriptome has little in common with the adult insects although larvae and adults feed on similar host resources. Proteomic studies revealed a sex-biased SG proteome, and that TSWV infection modulated the SG proteome in a sex-dependent manner. This is also true of the secretome. Surprisingly, the nonstructural TSWV protein, NSs was identified in female saliva. These studies are the first of their kind with the western flower thrips. In novel physiological studies, sex biased responses to dehydration and TSWV infection occurred relative to survival (females are larger and retain a larger percentage of their body mass as water). These studies also showed transcriptional shifts associated with glycogen metabolism and an increase in thrips feeding under desiccating conditions. Comparisons of infected and noninfected thrips further demonstrated that dehydration and infection changed thrips feeding patterns and altered survival. These studies were innovative and reveal parameters critical to addressing thrips and TSWV in a changing climate. Two salivary gland genes were transgenically-expressed in tomato. Experiments to measure thrips egg laying responses to expression of these genes were conducted. The results of these experiments were highly variable, in some cases due to loss of gene expression in the plants. Multiple generations of the transgenic plants are available for further work to quantify gene expression and protein production in these plants. Seeds were harvested, catalogued, and provided to the Rotenberg lab for additional studies (under permit). Finally, ten candidate proteins identified in female and/or male saliva secretions from TSWV-infected and/or non-exposed cohorts of thrips from our proteomic studies were selected to move forward for cloning into protein expression vectors designed for in planta, transient local expression and systemic expression in Nicotiana benthamiana and tomato. Four of these saliva proteins were successfully expressed in Nicotiana benthamiana as confirmed by western blot analysis, and these did not exhibit HR. These four proteins and TSWV proteins will be used for obtaining future preliminary data from assays designed to measure 'effector' activity, i.e., HR suppression and early defense responses (e.g. ROS). Future work is expected to evaluate promising 'effectors' in thrips feeding and oviposition bioassays to round out the phenotyping of these thrips saliva proteins. Aim 1. University of California Davis:Analysis of the salivary transcriptome has been refined and figures are under development. We expect to submit a manuscript in 2024 AIM 3: University of California Davis and North Carolina State University:Gene expression was detected in T0, T1 and T2 generations of tomato ('Moneymaker') transformed with the SG genes of interest. When gene expression was quantified in T3 and T4 generations we detected loss of expression in some plants. Measuring protein expression proved extremely difficult. With the loss of Ben-Mahmoud from the Ullman Laboratory, seed was collected and catalogued from all generations and sent, under permit, to the Rotenberg laboratory for future analysis. Functional analysis of 10 candidate proteins identified from saliva secretions of male and female (+/- TSWV) from our saliva protein analyses (Aim 2) were cloned into protein expression vectors intended for local (pGWB408 = Gateway cloning compatible binary vector for C-terminal fusion with 6xHis (CaMV35S promoter) and systemic (SPDK658(35S-PVX) = PVX T-DNA clone) expression of individual candidate proteins in planta (Rotenberg group). Four of these saliva proteins were successfully expressed as confirmed by western blot analysis, and these did not exhibit HR. These four proteins and TSWV protein constructs will be used for obtaining future preliminary data from assays designed to measure 'effector' activity, i.e., HR suppression and early defense responses (e.g. ROS). Future work is expected to evaluate promising 'effectors' in thrips feeding and oviposition bioassays to round out the phenotyping of these thrips saliva. University of Cincinnati:One paper describing the work with dehydration parameters has been published and a second is under review. The student working on this project received his M.S. and has taken a position with a pest control company.

Publications

• Type: Other Status: Other Year Published: 2023 Citation: Whitfield, A.E., and Rotenberg, D. Pests and Resistance. The biology of supervectors and superpests. 2023. Current Opinion in Insect Science 101060. Guest editorial introduction to the Pests and Resistance (June 2023) article collection of 9 invited author groups.
• Type: Journal Articles Status: Published Year Published: 2023 Citation: Maurastoni, M., Han, J., Whitfield, A.E., Rotenberg, D. 2023. A call to arms: novel strategies for thrips and tospovirus control. Current Opinion in Insect Science 57, 101033 doi.org/10.1016/j.cois.2023.101033
• Type: Journal Articles Status: Published Year Published: 2023 Citation: Bailey, S.T., Kondragunta, A., Choi, H.A., Han, J., Rotenberg, D., Ullman, D.E. and J.B. Benoit. 2023. Dehydration yields distinct transcriptional shifts associated with glycogen metabolism and increases feeding in the western flower thrips, Frankliniella occidentalis. Entomologia Experimentalis et Applicata. https://doi.org/10.1111/eea.13387
• Type: Journal Articles Status: Submitted Year Published: 2023 Citation: Bailey, S.T., Kondragunta, A., Choi, H.A., Han, J., Rotenberg, D., Ullman, D.E. and J.B. Benoit. 2024. Dehydration and tomato spotted wilt virus infection combine to alter feeding and survival parameters for the western flower thrips, Frankliniella occidentalis. Current Research in Insect Science (Submitted 12/21/23).
• Type: Journal Articles Status: Published Year Published: 2023 Citation: Benoit, Joshua B., Kevin E. McCluney, Matthew J. DeGennaro, and Julian AT Dow. "Dehydration Dynamics in Terrestrial Arthropods: From Water Sensing to Trophic Interactions." Annual Review of Entomology 68 (2023): 129-149.
• Type: Journal Articles Status: Published Year Published: 2023 Citation: Mutschler, M.A., Kennedy, G.G. and D.E. Ullman. 2023. Acylsugar-mediated resistance as part of a multilayered defense against thrips, orthotospoviruses, and beyond. Current Opinion in Insect Science.
• Type: Journal Articles Status: Published Year Published: 2023 Citation: Ordaz, N.A., Nagalakshmi, N., Boiteux. L.S., Atamian, H.S., Ullman, D.E. and S. P. Dinesh-Kumar. 2023. The Sw-5b NLR immune receptor induces earlier transcriptional changes in response to thrips-mediated inoculation of Tomato spotted wilt orthotospovirus compared to mechanical inoculation. Molecular Plant-Microbe Interactions. https://doi.org/10.1094/MPMI-03-23-0032-R
• Type: Journal Articles Status: Published Year Published: 2023 Citation: Yvon, M., German, T.L., Ullman, D.E., Dasgupta, R., Parker, M.H., Ben-Mahmoud, S., Verdin, E., Gognalons, P., Ancelin, Aurelie, Lai Kee Him, J., Girard, J., Vernerey, M., Fernandez, E., Filoux, D., Roumagnac, P., Bron, P., Michalakis, Y. and S. Blanc. 2023. The genome of a bunyavirus cannot be defined at the level of the viral particle but only at the scale of the viral population. PNAS 120 (No. 48) e2309412120.

Progress 09/01/21 to 08/31/22

Outputs
Target Audience:Scientific community: Rotenberg, D. Genome-enabled insights into thrips as crops pests and virus vectors, Super Vectors Symposium. Joint ESA-SEB / APS-CD Conference in San Juan, Puerto Rico. March 29, 2022. Rajarapu, S.P, Araujo, M., Whitfield, A.E., Benoit, J., Ullman, D.E. and Rotenberg, D. Mining the proteome of salivary glands for candidate modulators of thrips-virus-plant interactions in the Symposium: Omics tools and resources shed light on insect vector-pathogen interactions of agronomic significance. Joint ESA-SEB / APS-CD Conference in San Juan, Puerto Rico. March 28, 2022. Rajarapu, S.P., and Rotenberg, D. Sexually-dimorphic proteomic responses of Frankliniella occidentalis salivary glands to tomato spotted wilt virus infection. Entomological Society of America Conference, Denver, CO, Oct 31 - Nov 3, 2021 (in-person oral presentation). *SPR awarded NCSU Professional Development Award for Post Docs (Travel Award) Bailey, S.T., Kondragunta, A., Choi, H.A., Han, J., Rotenberg, D., Ullmann, D.E. and Benoit, J.B. Dehydration and viral infection yield similar stress that deplete glycogen and increase feeding in the Western Flower Thrips, Frankliniellaoccidentalis.. Arthropod Genomics Symposium, South Bend, Indiana. Changes/Problems:Loss of Sulley Ben-Mahmoud and lack of adequate funds to hire another postdoctoral researcher meant our focus needed to be limited to the work that could be completed with an undergraduate intern. Ullman will continue to work closely with the laboratory at North Carolina State to complete thrips oviposition studies and examine expression of two genes of interest. In addition, Ullman will transfer reagents and transgenic plants to North Carolina for continued work on Aim 3. What opportunities for training and professional development has the project provided?University of California Davis Ryan Packer, undergraduate intern, received training on working with thrips and orthotospoviruses from Ullman and learned RT qPCR in collaboration with the laboratories of Joanna Chui and Gitta Coaker. University of Cincinnati Samuel Bailey was trained in the Benoit laboratory on how to write scientific manuscripts and how to complete analyses of physiological data and transcriptomic data. North Carolina State University Swapna Priya Rajarapu (postdoc) competed for and awarded NCSU Professional Development Award for Post Docs (Travel Award used to present at Entomological Society of America Conference, Denver, CO, Oct 31 - Nov 3, 2021 (in-person oral presentation) Swapna Priya Rajarapu presented invited seminar: Stories from Invasive insects to virus vectors - Molecular plant-insect interactions. Department of Entomology, plant pathology and nematology. University of California-Davis, 2021 Swapna Priya Rajarapu participated in numerous postdoc professional development workshops on CV building, preparing effective job applications, and mentor-training of undergrads through the NCSU Graduate School and College of Agriculture and Life Sciences. How have the results been disseminated to communities of interest?Publications - Two peer-reviewed papers were published from the proteomic and physiological components of the project. Invited talks at Professional Society Meetings and Departmental Seminars What do you plan to do during the next reporting period to accomplish the goals?Work in the Benoit laboratory is complete. The focus will be on completing the final manuscript from Sam Bailey's research. UC Davis: 1. Screen transformed tomato lines for expression of genes of interest using RT-qPCR (September-December, 2022). 2. Produce seed from the most interesting lines (January-June, 2023). 3. Obtain permits and provide seed of transformed tomato to NCSU team (September 2022 to August 2023). 4. Complete analyses and publication of transcriptome. NCSU: AIM 3: Test how differentially expressed proteins from non-infected and infected WFT (western flower thrips) may vary in modulating plant defenses against the insect and the virus it transmits (TSWV) by phenotypic-screening of plants transiently-expressing SG-enriched proteins. Using tools and outputs produced from previous term (see above) the following tasks will be performed: Screen top saliva protein candidates for protein expression and plant defense expression in tomato using local expression system (binary agro plasmids) Clone top saliva protein candidates and a GFP (neg. control) into PVX-expression system (systemic) Screen PVX clones for expression of saliva proteins in tomato using vacuum infiltration system Perform replicated thrips feeding assays with best-performing PVX clones Perform thrips feeding assays on Arabidopsis mutant for saliva protein-interacting plant proteins Write manuscripts and repeat assays as needed

Impacts
What was accomplished under these goals? A salivary gland transcriptome was obtained for second instar larvae and adult males and females, infected and noninfected with TSWV was completed and analyzed. Selected several salivary gland enriched genes of interest for analysis of plant phenotype using transient expression in Nicotiana benthamiana. From these experiments, 2 genes of interest were selected for transformation into tomato (cv. Moneymaker). Transformants have been tested for presence and expression of the genes, and lines were selected and bred to the T4 generation. Quantification of gene expression is underway with RT-qPCR. Western flower thrips response to tomato transformed with the 2 genes of interest is being measured using oviposition assays. Preliminary results suggest expression of these proteins may enhance thrips oviposition. In addition, a salivary gland proteome and secretome was completed and analyzed for thrips adults (male and female), infected and non-infected. A manuscript describing the results was completed. Selected 16 thrips saliva-secreted proteins identified from Aim 2 research outputs with the criteria of: 1) present only in TSWV-infected treatments (male and female) or 2) sex-specific (male or female) or 3) shown to be effectors in other insect-plant interactions. Nine of the candidate saliva proteins have been successfully cloned into agrobacterium binary vector for transient expression assays. Construction of a GFP-expressing clone as an important negative control for plant expression assays - confirmed robust expression in plant tissue and no effect on defense gene panel. Four of these candidates were confirmed by western blot to be highly expressed in plant leaf tissue. Investigations to optimize other salivary proteins for expression in plant tissue are underway, as well as development of protein-protein assays. Assays to understand the effect of dehydration on thrips physiology and transmission of orthotospoviruses were completed and the first of two papers published. Aim 1. University of California Davis:First description of the salivary gland transcriptome of larval and adult male and female non-infected and infected western flower thrips (manuscript in preparation). From this work we selected several salivary gland enriched genes of interest for analysis of plant phenotype using transient expression in Nicotiana benthamiana. From these experiments, 2 genes of interest were selected for transformation into tomato (cv. Moneymaker). See Aim 3. AIM 2.North Carolina State University:The first description of thysanopteran (thrips) female and male (+/- TSWV) salivary gland proteome and saliva-secreted proteins of this global crop pest was completed and published. AIM 3: University of California Davis:Transformants identified from work in Aim 1 have been tested for presence and expression of the genes, and lines were selected and bred to the T4 generation. Quantification of gene expression is underway with RT-qPCR. Work began on assays to measure protein expression in transformed plants have been initiated using ELISA, and in collaboration with NC State (see their progress to date) Western blotting. See NC State progress for more information on understanding of protein-protein interactions. Western flower thrips response to tomato transformed with the 2 genes of interest is being measured using oviposition assays. Preliminary results suggest expression of these proteins may enhance thrips oviposition. North Carolina State University:Sixteen thrips saliva-secreted proteins identified from Aim 2 research outputs were selected using these criteria: 1) present only in TSWV-infected treatments (male and female) or 2) sex-specific (male or female) or 3) shown in Aim 3 to be effectors in other insect-plant interactions. Nine of the candidate saliva proteins have been successfully cloned into agrobacterium binary vector for transient expression assays. A GFP-expressing clone has been constructed as important negative control for plant expression assays. Using this clone, we confirmed robust expression in plant tissue and no effect on defense gene panel. At least 4 of these candidates confirmed by western blot to be highly expressed in plant leaf tissue and transient expression of two saliva proteins induced expression of plant defense genes in preliminary tests. In Arabidopsis protein-protein interaction assays several critical plant proteins (chloroplast) interacted with one saliva protein, while a second saliva protein interacted with a different set of proteins. We have been working with UC Davis to optimize detection of tomato transgenic expression of two saliva proteins. Work is underway to clone the 4 saliva protein candidates into a PVX viral vector for systemic expression in plants in N. benthamiana and tomato to enable thrips performance/phenotyping assays. University of Cincinnati:Samuel Bailey completed his Masters Degree and wrote his thesis and took a position at a pest control company. One publication from his thesis was published.

Publications

• Type: Journal Articles Status: Published Year Published: 2022 Citation: Bailey, S.T., Kondragunta, A., Choi, H.A., Han, J., Rotenberg, D., Ullman, D.E. and J.B. Benoit. 2022. Dehydration and infection elicit increased feeding in the western flower thrips, Frankliniella occidentalis, likely triggered by glycogen depletion. Biorxiv.org.
• Type: Journal Articles Status: Published Year Published: 2022 Citation: Rajarapu, S.P., Ben-Mahmoud, S., Benoit, J.B., Ullman, D.E., Whitfield, A.E., and D. Rotenberg*. 2022. Sex-biased proteomic response to tomato spotted wilt virus infection of the salivary glands of Frankliniella occidentalis, the western flower thrips. Insect Biochemistry and Molecular Biology 149 103843.
• Type: Theses/Dissertations Status: Other Year Published: 2022 Citation: Bailey, Samuel. "Dehydration and infection elicit increased feeding in the western flower thrips, Frankliniella occidentalis, likely triggered by glycogen depletion." Masters dissertation, University of Cincinnati, 2022. https://doi.org/10.1111/eea.13387
• Type: Journal Articles Status: Published Year Published: 2022 Citation: Benoit, J.B., Lahond�re, C., Attardo, G.M., Michalkova, V., Oyen, K., Xiao, Y. and Aksoy, S., 2022. Warm blood meal increases digestion rate and milk protein production to maximize reproductive output for the tsetse fly, Glossina morsitans. Insects, 13(11), p.997.
• Type: Journal Articles Status: Published Year Published: 2022 Citation: Ordaz, N.A., Nagalakshmi, N., Boiteux. L.S., Atamian, H.S., Ullman, D.E. and S. P. Dinesh-Kumar. 2022. The Sw-5b NLR immune receptor induces earlier transcriptional changes in response to thrips-mediated inoculation of Tomato spotted wilt orthotospovirus compared to mechanical inoculation. BIORXIV/2022/507022.

Progress 09/01/20 to 08/31/21

Outputs
Target Audience:Scientific Community: Ullman, UC Davis 1) Invited Presentation, Aggie Women in Entomology, Department of Entomology, Texas A&M, September 30, 2020 2)Invited Presentation: Larry Vanderhoef Memorial Lecture, National Chung Hsing University, November 18, 2020 3) Invited Presentation: Specialty Crops Committee, February 21, 2021 Rotenbert- North Carolina State University: Postdoc (Rajarapu) oral presentation at professional society: Rajarapu, S.P., and Rotenberg, D. "Sexually-dimorphic proteomic responses of Frankliniella occidentalis salivary glands to tomato spotted wilt virus infection." Entomological Society of America Conference, Denver, CO, Oct 31 - Nov 3, 2021 (in-person oral presentation). Postdoc (Rajarapu) oral presentation at professional society meeting: "Identifying TSWV modulated proteins in the salivary glands and saliva of western flower thrips, Franklinella occidentalis." Southeastern Branch of the Entomological Society of America, virtual meeting, March 2021. Invited seminar (Rotenberg): Molecular interactions between thrips and tospoviruses: Advances in defining determinants of thrips vector competence. Department of Biological Sciences, Seminar Series, University of Cincinnati, OH, March 29, 2021. Invited professional society symposium talk (Rotenberg): It takes a lot of guts: characterizing the early response to tomato spotted wilt virus infection in larval thrips. The Entomological Society of America Annual Meeting, Plant-Insect Ecosystems Section Symposium: Transmission Ecology of Vector-borne Phytopathogens, St. Louis, MO, Nov 20, 2019. Community Audiences: Ullman: Community Engaged Learning Fellowship, Art, Science and the World of Insects, Bringing Science and Art Together Using a Community Engagement Strategy. July 30, 2021. Changes/Problems:University of California Davis: The COVID-19 pandemic and associated lockdown and quarantine significantly impacted our progress towards our goals. When were allowed back on campus, summer 2020, our isolates had to be started again from materials we froze. The thrips colony had to be built up to a useable number of insects. Planting of the transgenic plants for seed production and selection had to be started, as this was on hold during the lockdown. Ben-Mahmoud's J1 visa could not be renewed and ended June 30, 2021. In anticipation of this fact, he applied for permanent residency in 2018, but he didn't learn that this was denied until February 2021. He appealed this decision, but the appeal was pending when the J1 visa ended, so June 30, 2021 was his last day working in the lab. This was a very unfortunate time to be required to leave, as he was analyzing data, and preparing to do many assays on plant defenses, and thrips responses to the plants transformed with SG-enriched genes. Given the funding remaining in the California budget, it has not been possible to replace Sulley. He plans to assist in writing the work he completed, but this was a blow to our progress. We added a student laboratory assistant to the lab in 2020, and he has learned to do the oviposition assays we use to assess thrips responses to the transgenic plants expressing thrips salivary gland genes. Losing Sulley, a key member of our team, has definitely slowed our progress. The funding remaining at UC Davis is not really adequate to bring in a project scientist or postdoctoral scholar to replace Sulley. Instead, these funds will be used to work in collaboration with the Rotenberg and Benoit laboratories to complete Aim 3 objectives Sulley has agreed to participate in writing of the publication about the transcriptomic work (Aim 1), and Ullman and a student intern are completing oviposition and feeding experiments to assess thrips responses to the transgenic plants. Finally, Ullman will work with Sam Bailey and Josh Benoit to complete analysis of tomato responses to uninfected and infected WFT. University of Cincinnati: The COVID-19 pandemic and associated lockdown and quarantine significantly impacted our progress towards our goals. We were able to maintain the thrips, but from March 2020 until July 2020 there have been specific delay. These range from reduced personnel to delays in orders). We anticipate that the project goals at University of Cincinnati will be complete in the NCE. All outreach events have been cancelled through 2020, but we anticipate that these will resume in 2021. North Carolina State University: The COVID-19 pandemic and associated lockdown and quarantine significantly impacted our progress towards our goals. On March 19, 2020, NC State University shutdown all research activities and limited access to facilities to approved, mandatory employees taking care of livestock, e.g., plants, insects and obligate microbes/viruses. Rotenberg's personnel on this project teleworked entirely from home (data analysis and writing) until May 22, 2020, at which time research started up at 35% capacity, allowing personnel to work socially-distant in shifts with restricted/no access to some of her growing facilities. The research shut-down and limited research re-start compromised potential progress made on Aim 2. In June 2021, Rotenberg's postdoc on this project experienced a significant, unexpected and urgent matter that required international travel and an extended stay in her country of origin. Major slowdowns with travel visa approvals due to the pandemic resulted in her postdoc returning in September 2021. This absence impacted research progress (bench-work) made on Aim 2 and 3. Nonetheless, her postdoc made significant progress on writing her first-author manuscript for Aim 2, and has since made significant progress on saliva protein cloning and in planta analyses. What opportunities for training and professional development has the project provided?University of California Davis Sulley Ben Mahmoud has: 1. Reporting and presentations to project team: Recurrent Zoom 'lab meetings' with the other project PI's and staff/students on this collaborative project. 2. Learned new techniques for measuring protein expression in transgenic plants. 3. Mentored an undergraduate and graduate student in the laboratory, teaching analysis with RT-qPCR and gene silencing techniques, and how to do biological experiments with thrips and TSWV. 4. Participated in remote seminars in Plant Pathology and Entomology. University of Cincinnati Samuel Bailey has: 1. Reporting and presentations to project team: Recurrent Zoom 'lab meetings' with the other project PI's and staff/students on this collaborative project. 2. Developed novel techniques to assay feeding in thrips. 3. Has developed skills on RNA-seq analysis that has been used on thrips and tomato datasets. 4. Mentored two undergraduate students that are expected to be involved as an author on the manuscript. North Carolina State University Swapna Priya Rajarapu has: Reported and presented to project team: Recurrent Zoom 'lab meetings' with the other project PI's and staff/students on this collaborative project. Developed techniques for thrips saliva collection and protein isolation Developed and sharpened skills in gene cloning and molecular/functional analyses Competed for and awarded a NCSU Professional Development Award for Postdocs (2021) and used the funds to travel to Denver, CO to present her research findings at the Annual Entomological Society of America Conference (ESA 2021). Fully participated in professional development workshops offered at the ESA 2021 Annual Conference, including those focused on writing (resume) and oral communication skills, navigating and embracing diversity, teaching technologies Served as a judge of student performance at ESA 2021 (poster and presentation judging). How have the results been disseminated to communities of interest?Too early for dissemination. We expect that three-four of the major studies will be completed and submitted for publication in 2022. What do you plan to do during the next reporting period to accomplish the goals?University of California Davis: Work with Benoit and Rotenberg to complete analysis of the salivary gland transcriptome and prepare it for publication. Perform experiments to determine plant (tomato) defense response to WFT salivary protein products and determine impact on WFT physiology in collaboration with Rotenberg and Benoit. Complete analysis of transgenic plants with regards to transgene and protein expression in collaboration with Rotenberg. University of Cincinnati: Work with Ullman and Rotenberg to complete analysis of the salivary gland transcriptome and prepare it for publication. Complete thrips dehydration project. North Carolina State University: Work with Ullman and Benoit to finalize manuscript for publication of salivary gland proteomes Clone and transiently express up to 10 candidate saliva proteins of interest based on their response to TSWV, sex and/or functional roles in other insect pest-plant interactions in preparation for thrips feeding bioassays in collaboration with Ullman

Impacts
What was accomplished under these goals? General: A salivary gland transcriptome and proteome was completed for second instar larvae (transcriptome only), and adult males and females, infected and noninfected with TSWV. Adult saliva proteins were sequenced to reveal a diverse array of secreted proteins with known roles in other insect-plant interactions, uncharacterized proteins, and apparently F. occidentalis-specific proteins. The identities of the saliva-secreted proteins confirmed findings from the salivary gland tissue proteome, including proteins predicted to be secreted. One gene of interest encoding a secreted protein was transiently-expressed locally in plants, with molecular confirmation of protein expression in planta. This protein interacted directly with several plant proteins in a robust yeast-2-hybrid screen. Tomato Moneymaker was transformed with two other genes of interest that encode thrips secreted salivary proteins. The transformed plants have been grown to the T3 generation, and preliminary thrips bioassay results suggest that at least one transgenic line enhances thrips performance (oviposition) in replicated assays. A novel thrips assay was developed to examine water balance dynamics in non-infected and TSWV-infected males and females to determine if virus infection status alters dehydration. Aim 1.University of California Davis: A salivary gland transcriptome has been completed for second instar larvae, and adult males and females, infected and noninfected with TSWV. Preliminary analyses show that thrips SG-enriched transcripts are differentially expressed in response to development and sex. While differential expression of some genes occurred relative to TSWV-infection status, the impact of virus infection was less extensive than expected. We expect that this study will be completed in early 2022 and submitted for publication during that time. AIM 2. North Carolina State University Saliva was collected from several cohorts of non-exposed and TSWV-infected male and female thrips. Proteins were concentrated and sent to Duke Center for Genomics and Computational Biology for qualitative analysis by 1-dimensional ultra-performance liquid chromatography-tandem mass spectrophotometry (1D UPLC-MS/MS). Secreted saliva proteins were identified using a sequence database of predicted peptide sequences generated for our published Frankliniella occidentalis official gene set (OGS v1.0; Rotenberg D, et al. 2019; Ag Data Commons, and TSWV protein references using Mascot search engine. A total of 704 proteins were identified from the secreted saliva of males and females irrespective of the infection status. Among these, 16% were identified in the salivary gland tissue proteome sequenced in year 1 of the project and 6% proteins were identified as enriched in the salivary glands relative to the whole body as per our previously published study (Rotenberg et al 2020). Proteins with "Generation of precursor metabolites and energy", "Ribosome", "Peptidase activity", "Carbohydrate metabolic process", and "hydrolase activity" Gene Ontology terms were enriched in the secreted saliva of thrips. The secreted proteins were predicted to be localized to all cell organelles with cytoplasmic, extracellular, and mitochondrial proteins as the most abundant. Although most of the saliva proteins (42%) were shared between males and females regardless of infection status, several proteins unique to virus exposure or sex were also identified. We have completed a draft manuscript that describes the salivary gland and saliva proteomes of F. occidentalis female and male thrips in response to TSWV to be submitted for peer review in January 2022. AIM 3: University of California Davis: We identified a set of salivary proteins of interest (year one). Based on these findings, we cloned and sequence verified selected WFT SG-enriched transcripts and worked with the Plant Transformation Facility, at UC Davis to transform tomato Moneymaker. Transformants have been reared to the T3 generation, and oviposition experiments with WFT suggest that at least one gene of interest may suppress plant defenses. These experiments are being replicated, and experiments to measure protein expression, and impacts on plant defenses were initiated. University of Cincinnati: We have completed the first study to examine the water balance parameters of thrips, which consisted of examining how viral infection could impact this process. This consisted of measuring dehydration stress resistance in males and females without infection and developing a feeding assay to establish how dehydration impacts feeding. RNA-seq was utilized to examine how dehydration alters the thrips. Lastly, we examined if infection status altered dehydration and how dehydration-infection dynamics impact thrips feeding rates. We expect that this study will be completed in early 2022 and submitted for publication during that time. North Carolina State University: A subset of candidate saliva protein sequences (from Aim 2) were cloned into a His-tagged binary vector expression plasmid for transient expression (local) of candidate saliva proteins for interest in Nicotiana benthamiana. To date, in planta transient expression of one protein of interest was confirmed by Western blot. A yeast-2-hybrid (Y2H) screen of an Arabidopsis thaliana cDNA library and the candidate saliva protein of interest using stringent selection of putative interactors, followed by confirmatory Y2H with putative interactors and the candidate saliva protein of interest revealed 17 'true' interactors, a few of which were predicted bioinformatically (STRING) to interact with pathway members implicated in plant defense. A second protein of interest that was determined to influence thrips performance via tomato transgenics (see Aim 3: UC-Davis) was also subjected to the same Y2H screen with the A. thaliana cDNA library. Two putative interactors were identified in the screen. We are currently developing experiments to determine the effect of the candidate saliva proteins on plant defense gene expression and plant biochemical activity, and we hope to continue functional analyses of saliva proteins of interest in plant assays.

Publications

• Type: Journal Articles Status: Published Year Published: 2020 Citation: Dorith Rotenberg, Aaron A. Baumann, Sulley Ben-Mahmoud, Olivier Christiaens, Wannes Dermauw, Panagiotis Ioannidis, Chris G.C. Jacobs, Iris M. Vargas Jentzsch, Jonathan E. Oliver, Monica F. Poelchau, Swapna Priya Rajarapu, Derek J. Schneweis, Simon Snoeck, Clauvis N.T. Taning, Dong Wei, Shirani M. K. Widana-Gamage, Daniel S.T. Hughes, Shwetha C. Murali, Sam Bailey, Nicolas E. Bejerman, Christopher J Holmes, Emily C. Jennings, Andrew J. Rosendale, Andrew Rosselot, Kaylee Hervey, Brandi A. Schneweis, Sammy Cheng, Christopher Childers, Felipe A. Sim�o, Ralf G. Dietzgen, Hsu Chao, Huyen Dinh, HarshaVardhan Doddapaneni, Shannon Dugan, Yi Han, Sandra L. Lee, Donna M. Muzny, Jiaxin Qu, Kim C. Worley, Joshua B. Benoit, Markus Friedrich, Jeffery W. Jones, Kristen A. Panfilio, Yoonseong Park, Hugh M. Robertson, Guy Smagghe, Diane E. Ullman, Maurijn van der Zee, Thomas Van Leeuwen, Jan A. Veenstra, Robert M. Waterhouse, Matthew T. Weirauch, John H. Werren, Anna E. Whitfield, Evgeny M. Zdobnov, Richard A. Gibbs, Stephen Richards. 2020. Genome-enabled insights into the biology of thrips as crop pests. BMC Biol 18, 142 (2020). https://doi.org/10.1186/s12915-020-00862-9.
• Type: Journal Articles Status: Published Year Published: 2021 Citation: Rajarapu, S.P., Ullman, D.E., Uzest, M., Rotenberg, D., Ordaz, N.A., and Whitfield, A.E. 2021. Plant-Virus-Vector-Interactions IN Virology, Carla Saleh and Felix Rey, eds. ISTE Sciences, pp. 227-288, Wiley, ISBN: 9781789450231, 364 pp.
• Type: Journal Articles Status: Published Year Published: 2021 Citation: Davies B, Rosendale AJ, Gantz JD, Lee RE, Denlinger DL, Benoit JB. Cross-tolerance and transcriptional shifts underlying abiotic stress in the seabird tick, Ixodes uriae. Polar Biology. 2021 Jun 8:1-1
• Type: Journal Articles Status: Published Year Published: 2021 Citation: Oyen KJ, Croucher L, Benoit JB. Tonic Immobility Is Influenced by Starvation, Life Stage, and Body Mass in Ixodid Ticks. Journal of Medical Entomology. 2021 May;58(3):1030-40.
• Type: Journal Articles Status: Published Year Published: 2021 Citation: Fieler AM, Rosendale AJ, Farrow DW, Dunlevy MD, Davies B, Oyen K, Xiao Y, Benoit JB. Larval thermal characteristics of multiple ixodid ticks. Comparative Biochemistry and Physiology Part A: Molecular & Integrative Physiology. 2021 Jul 1;257:110939.
• Type: Journal Articles Status: Published Year Published: 2021 Citation: Ajayi OM, Gantz JD, Finch G, Lee Jr RE, Denlinger DL, Benoit JB. Rapid stress hardening in the Antarctic midge improves male fertility by increasing courtship success and preventing decline of accessory gland proteins following cold exposure. Journal of Experimental Biology. 2021 Jan 1.

Progress 09/01/19 to 08/31/20

Outputs
Target Audience:Scientific Community: Ullman 1) Invited Presentation: Opening Plenary address for the XI International Symposium on Thysanoptera and Tospoviruses in Kunming, China (September 2019). 2) Invited Presentation: Impact of Tomato Spotted Wilt Virus Infection on Transcript Expression in the Salivary Glands of Frankliniella occidentalis and Potential Effects on Plant Defense, Plenary Vector-Virus Session, XI International Symposium on Thysanoptera and Tospoviruses in Kunming, China (September 2019). Sulley Ben-Mahmoud: 1. Invited Presentation: Functional Analysis of Western Flower Thrips Salivary Gland Genes, National Meeting of the Entomological Society of America (November 2019). Dorith Rotenberg (note, these invited talks were cancelled due to COVID-19 lockdowns): 1. Genome-enabled advances in the characterization of molecular interactions between Frankliniella occidentalis and tomato spotted wilt virus. University of Georgia, Department of Entomology, Athens, GA. Annual Lund Week Entomology Seminar - H.O. Lund Club, Entomology Graduate Student Club nominated and selected seminar speaker, Scheduled for April 17, 2020. Cancelled due to the COVID-19 pandemic. (virtual talk rescheduled for October 19, 2020) 2. Genome-enabled advances in the characterization of molecular interactions between Frankliniella occidentalis and tomato spotted wilt virus. Oklahoma State University, Department of Entomology and Plant Pathology, Stillwater, OK. Scheduled for April 8, 2020. Cancelled due to the COVID-19 pandemic. 3. Genome-enabled advances in the characterization of molecular interactions between Frankliniella occidentalis and tomato spotted wilt virus. Colorado State University, Department of Bioagricultural Sciences and Pest Management, Fort Collins, CO. Scheduled for March 11, 2020. Cancelled due to the COVID-19 pandemic. Community Audiences: Joshua Benoit: Benoit, J. B. Mosquito, ticks, and disease vectors of Ohio. Fernald Nature Preserve, US Department of Energy. Hamilton, OH. Benoit, J. B. Mosquito, ticks, and other disease vectors: Ohio natives that everyone should know. University of Cincinnati Center for Field Studies, Harrison, OH. Changes/Problems:University of California Davis: The COVID-19 pandemic and associated lockdown and quarantine significantly impacted our progress towards our goals. Ben-Mahmoud and Ullman teleworked entirely from home conducting data analysis and writing until June 1. At that time we were allowed limited access back to our laboratory. We were allowed to add a student laboratory assistant to the lab in August and have been inching back towards more normal operations. We are working hard to make up for lost time and expect to able to fulfill our objectives in a timely fashion barring any future lockdowns or quarantines. North Carolina State University: Like most researchers around the U.S., the Rotenberg lab dramatically reduced research operations due to concerns with the COVID-19 pandemic. On March 19, 2020, NC State University shutdown all research activities and limited access to facilities to approved, mandatory employees taking care of livestock, e.g., plants, insects and obligate microbes/viruses. Rotenberg's personnel on this project teleworked entirely from home (data analysis and writing) until May 22, 2020, at which time research started up at 35% capacity, allowing personnel to work socially-distant in shifts with restricted/no access to some of her growing facilities. The research shut-down and limited research re-start compromised potential progress made on Aim 2, namely saliva collection and proteomic analysis (DUKE Proteomics & Metabolomics Core Facility shut-down their services during this period as well). University of Cincinnati: Sam Bailey has been an excellent addition to the team, but we have been delayed in the establishment of a thrips colony. Space was required to house thrips has been established and the colony is now proliferating. The main focus will be performing the initial stress tolerance assays and then examining these in relation to infection. Productivity in 2020 has been reduced due to coronavirus restrictions, but substantial progress has occurred. What opportunities for training and professional development has the project provided?University of California Davis Sulley Ben Mahmoud has: 1. Reporting and presentations to project team: Recurrent Zoom 'lab meetings' with the other project PI's and staff/students on this collaborative project. 2. Learned new techniques for salivary gland dissection, RNA extraction, and transient expression of salivary gland transcripts. 3. Mentored a graduate student in the laboratory as she learns RT-qPCR and gene silencing techniques. 4. Provided an invited seminar to the Entomology Society of American. 5. Participates in remote seminars in Plant Pathology and Entomology. North Carolina State University: Priya Rajarapu (postdoctoral scholar), was engaged in several types of training and professional development opportunities listed below: 1. Reporting and presentations to project team: Recurrent Zoom 'lab meetings' with the other project PI's and staff/students on this collaborative project. 2. Presented at Campus-wide symposium: "Sex-specific salivary gland proteome of a thrips vector" presented at Emerging Plant Disease - Global Food Security Cluster Symposium: Outbreaks: Tackling Emerging Plant Diseases That Threaten Food Security, NCSU, Raleigh, NC, Jan 10, 2020. 3. NC State Office of Postdoctoral Affairs Research Symposium: Submitted a 5-minute lightning presentation entitled "New frontiers in insect vector transmission of plant viruses: The salivary gland proteome of western flower thrips in response to tomato spotted wilt virus infection" scheduled for May 29, 2020 (Canceled due to COVID19). 4. Undergraduate mentoring: Mentored and supervised the undergraduate student, Katy Cernava, enrolled in ENT493: Special Problems in Entomology to conduct a research project to determine the effect of resorcinol on adult diet consumption. 5. Writing and peer-review: Participated in a Writing Accountability Group (WAG) with other NCSU postdocs (Facilitated by Dr. Dorith Rotenberg) launched in April 2020 during the NCSU shutdown in response to COVID19. 6. Bolstering critical-thinking skills and comradery: Participated in a Postdoc/Staff-only Journal Club in Vector-Virus Biology organized by a fellow postdoc in Dr. Anna Whitfield's lab and launched in April 2020. 7. Knowledge-building and awareness of current studies in human/vertebrate vector biology: Attended the virtual Vector Biology Seminar Series II (July - September 2020) organized by Drs. Dana Shaw and Kelly Breyton, Washington State University. University of Cincinnati: Samuel Baily (Graduate student): 1. Learned new bioinformatic techniques to examine RNA-seq data sets. 2. Multiple courses on professional development. 3. In relation to arthropod-plant interactions, Bailey has mastered thrips husbandry and can now begin his projects that are the core of his Master's project on thrips physiology. How have the results been disseminated to communities of interest?Too early for dissemination. What do you plan to do during the next reporting period to accomplish the goals?University of California Davis: Submit RNA samples for RNA-seq library preparation and sequencing. Perform experiments to determine plant (tomato) defense response to WFT salivary protein products and determine impact on WFT physiology. Perform further cloning of WFT-SG transcripts for transient expression in-planta given results of RNA-seq analysis. North Carolina State University: Perform TSWV acquisition experiments for saliva collection from larval and adult male and female thrips to determine the composition and abundance of secreted proteins in response to virus infection. Compare SG proteomic (Aim 2) and transcriptomic data (Aim 1) to determine overlap for selection of primary candidates for effector functional analysis in thrips performance tests (Aim 3 with D. Ullman). Perform transient, localized expression screens of candidate SG genes in model (Nicotiana benthamiana) and host (Lycopersicum esculentum) plants using binary expression vectors and share elite candidates with Ullman group for PVY vector-assisted transient, systemic expression in plants to enable thrips performance, fitness and physiological tests. Manuscript preparation University of Cincinnati: Complete analysis of tomato plants (see RNAseq analysis above). Perform initial RNA-seq studies from sexes and developmental stages (both infected and uninfected SGs). Perform baselines stress tolerance of uninfected thrips. Examine if TSWV impacts thrips stress tolerance. Examine nutritional reserve levels uninfected thrips. Determine if TSWV impacts nutritional reserve status. Establish baselines for thrips behavior (circadian, etc.). Establish if infection alters thrip behavior such as circadian rhythm changes.

Impacts
What was accomplished under these goals? General: We completed salivary gland (SG) dissections for transcriptomics and proteomics, developed high quality RNA extraction and protein preparation methods, transiently expressed candidate thrips SG transcripts and identified interesting phenotypes; as well as, producing tomato plants transformed with the two most promising candidates. A salivary gland proteome has been completed for all stages and noninfected and infected thrips examined. Thrips colonies are established for physiological studies at UNC. Aim 1. University of California Davis: Dissections of SGs of TSWV-infected and non-infected larvae, and male and female adults are completed (4 biological replicates, 50 pooled SGs/rep from second instar and male and female adult stages). As TSWV doesn't infect the first instar's salivary glands, we eliminated study of this stage. A modified TRIzol® protocol worked well to yield quality RNA in the quantity needed for RNAseq. COVID-19 pandemic limitations delayed RNAseq studies. A test sample of adult RNA will be processed next month. If successful, we will extract RNA and submit all samples for RNA library preparation, sequencing and analysis by Joshua Benoit's laboratory (UNC). RT-qPCR showed virus infection altered expression of at least one SG- enriched transcript in adult females. To explore expression of these genes in multiple WFT stages, we examined expression in whole bodies of all stages, non-infected and infected. These experiments showed that expression in organs outside of the salivary glands occurs in response to virus infection and masks changes observed in dissected SGs. The difference in expression of SG-enriched transcripts in SG tissues versus whole body supports our tissue specific approach in determining how virus infection may alter gene expression. University of Cincinnati: Salivary gland associated genes have been identified and published. Bioinformatics pipelines using implementation of advanced expressional analyses, including weighted correlation network analysis (WGCNA) to ensure that all analyses are thorough and complete. Infected and non-infected samples can now be analyzed following Illumina sequencing. Aim 2. North Carolina State University: Building on our proteomic analysis of SG proteins identified from non-exposed adult thrips (year 1), we performed repeated factorial experiments to determine the effect of TSWV infection (+/- virus exposure as larvae) on the proteomes of female and male SGs. First instar larvae were fed on TSWV-infected and healthy Datura stramonium leaves for a 24-hour acquisition access period, and larval cohorts with high infection rates (> 70%, based on real-time quantitative reverse transcription-PCR) were moved to green bean pods to develop to adulthood. Salivary glands (SG) from young infected and non-infected adults were dissected to achieve four biological replicates (i.e., independent acquisition experiments), each replicate consisting of a pool of female (100 pairs) and male (200 pairs) SGs. The samples (16 in total) were sent to the Duke Center for Genomic and Computational Biology, Proteomics and Metabolomics Core Facility for protein extraction, sample preparation and quantification by 1-dimensional ultra-performance liquid chromatography-tandem mass spectrophotometry (1D UPLC-MS/MS). Salivary gland proteins were identified using a sequence database of predicted peptide sequences generated for our published Frankliniella occidentalis official gene set (OGS v1.0; Rotenberg D, et al. 2019; Ag Data Commons (Database). https://doi.org/10.15482/USDA.ADC/1504029). We used the MSStats R Bioconductor package and bioinformatics analyses to identify and annotate differentially- expressed proteins. In total, over 2,500 non-redundant proteins were captured from the SGs of both sexes with approximately 3.5% having no significant matches to known proteins (likely thrips-specific) or annotated as uncharacterized proteins in other organisms. Principal components analysis revealed that sex was the largest driver of the variation in expression levels (normalized abundance) of proteins in the datasets. Proteomic response to TSWV infection was sexually-dimorphic, in that virus infection significantly perturbed a unique set of proteins between the sexes when comparing infected individuals to their non-infected counterparts (Padj < 0.05). Of the TSWV-responsive SG proteins, 9% and 5% contained predicted structural features indicative of secreted proteins in females and males, respectively. A subset of candidate sequences are being cloned into binary vector expression plasmids for transient expression of the SG peptides in Nicotiana benthamiana or Lycopersicum esculentum to characterize plant responses. Experiments are also under way to i) optimize biochemical and molecular analyses of canonical plant defense enzymes to enable quantification of these proteins in response to expression of SG candidates in planta, and ii) to collect saliva from infected and non-infected larvae, males and females to sequence secreted proteins for comparisons with SG-tissue proteomes and structural predictions. AIM 3: University of California Davis: We identified a set of salivary proteins of interest (year one). Based on these findings, we cloned and sequence verified selected WFT SG-enriched transcripts into the plant expression vectors: pK2GW7, pDU12.0310, and pYL254 and pSPDK2483. Using vectors pYL254 and pSPDK2483, we built constructs to transiently express WFT proteins of selected SG-enriched transcripts (predicted to be secretory proteins) in Nicotiana benthamiana. Using Agrobacterium-mediated transient expressions two WFT SG-enriched constructs (SG114 and SG053) elicited a phenotypic response on leaves of N. benthamiana that was not present in empty vector controls. Two pDU12.0310:WFT-SG constructs (containing SG053 and SG114 inserts) were submitted to the Plant Transformation Facility, at UC Davis for tomato Moneymaker transformation. After multiple submissions, transformations were completed for SG114 (10) and SG053 (3). We confirmed presence of both inserts and are quantifying RNA expression, protein expression, and are propagating the plants. Experiments have begun to determine impact SG114 and SG053 on plant defenses and WFT biology. Unfortunately, transient expression in N. benthamiana is not useful for assessing WFT biology because they do not feed or thrive on this plant species. University of Cincinnati: Progress on has been delayed due to COVID-19 restrictions. We now have thrips cultures at the University of Cincinnati and have begun preliminary studies on stress tolerance and behavior. The next goal will be to examine these differences in relation to infection status. During previous work, Ullman conducted RNAseq on tomato isolines with and without the Sw-5 gene subjected to four treatments: non-infected WFT, TSWV-infected WFT, TSWV mechanical inoculations, and mechanical inoculation with noninfected tomato sap (mock inoculation) (3 biological replicates). We have been conducting analyses of this RNAseq data set to reveal plant responses to non-infected thrips and to determine if these responses are altered when the thrips are infected with TSWV, and whether the presence of the Sw-5 gene (resistance gene for TSWV) influences these responses. We expect this analysis to help guide the defense genes we may explore in tomatoes transformed with thrips SG-enriched genes.

Publications

• Type: Journal Articles Status: Awaiting Publication Year Published: 2020 Citation: Rotenberg, D.*, Baumann, A.A., Ben-Mahmoud, S., Christiaens, O., Dermauw, W., Ioannidis, P., Jacobs, C.G.C., Vargas Jentzsch, I.M., Oliver, J.E., Poelchau, M.F., Rajarapu, S.P., Schneweis, D.J., Snoeck, S., Taning, C.N.T., Wei, D., Widana-Gamage, S.M.K., Hughes, D.S.T., Murali, S.C., Bailey, S., Bejerman, N.E., Holmes, C.J., Jennings, E.C., Rosendale, A.J., Rosselot, A., Hervey, K., Schneweis, B.A., Cheng, S., Childers, C., Sim�o, F.A., Dietzgen, R.G., Chao, H., Dinh, H., Doddapaneni, H., Dugan, S., Han, Y., Lee, S.L., Muzny, D.M., Qu, J., Worley, K.C., Benoit, J.B., Friedrich, M., Jones, J.W., Panfilio, K.A., Park, Y., Robertson, H.M., Smagghe, G., Ullman, D.E., Van Der Zee, M., Van Leeuwen, T., Veenstra, J.A., Waterhouse, R.M., Weirauch, M.T., Werren, J.H., Whitfield, A.E., Zdobnov, E.M., Gibbs, R.A., and Richards, S. 2020. Genome-enabled insights into the biology of thrips as crop pests. BMC Biology, in press.
• Type: Journal Articles Status: Published Year Published: 2020 Citation: Adjacent publications: The grant was acknowledged in the following non-thrips projects as techniques we have been developing for thrips studies were used for other projects by the PI and graduate students. Jennings, E. C., Korthauer, M. W., Hendershot, J. M., Bailey, S. T., Weirauch, M. T., Ribeiro, J. M., & Benoit, J. B. (2020). Molecular mechanisms underlying milk production and viviparity in the cockroach, Diploptera punctata. Insect Biochemistry and Molecular Biology, 103333.
• Type: Journal Articles Status: Awaiting Publication Year Published: 2020 Citation: Adjacent publications: The grant was acknowledged in the following non-thrips projects as techniques we have been developing for thrips studies were used for other projects by the PI and graduate students. Ajayi, O. M., Eilerts, D. F., Bailey, S. T., Vinauger, C., & Benoit, J. B. (2020). Do Mosquitoes Sleep?. Trends in Parasitology. In press

Progress 09/01/18 to 08/31/19

Outputs
Target Audience:Presentations have been given to scientists and practitioners with an interest in thrips as vectors. Ullman has given three presentations to audiences with special interests in insect vectors of plant pathogens: 1) seminar, BGPI (Biologie et Génétique des Interactions Plante-Parasite --Biology and Genetics of Plant-Parasite Interactions) INRA Montpellier, France; 2) plenary address, Rencontres Virologie Végétale 2019 (most important French meeting of plant virologists, held every two years); and, seminar, Universite de Lyon. Benoit has provided two presentations at local community events: Benoit, J. B. Mosquito, ticks, and disease vectors of Ohio. Fernald Nature Preserve, US Department of Energy. Hamilton, OH. Benoit, J. B. Mosquito, ticks, and other disease vectors: Ohio natives that everyone should know. University of Cincinnati Center for Field Studies, Harrison, OH. Rotenberg has presented the project over arching goal and thrips genome-enabled preliminary findings as part of a contributed talk to international audience of the professional society, International Society-Molecular Plant Microbe Interactions (IS-MPMI): Molecular interactions between tospoviruses and thrips vectors. International Society - Molecular Plant Microbe Interactions, XVIII Congress, Satellite Meeting: Dynamics and Mechanisms of Insect-Transmitted Pathogens, Glasgow, Scotland, UK, July 14, 2019. Changes/Problems:An initial challenge was recruitment of a postdoctoral scholar (NC State) and a graduate student (University of Cincinnati). However, excellent people have been hired into these positions. Great progress has been made at all three collaborating institutions. The experiments leading to transcriptomic and proteomic studies have proceeded well, with the major challenge being dissections of salivary glands from first instar larvae. At this life stage the insects are very small; however, progress has been made in this arena and we expect dissections to now proceed smoothly. At UC Davis, a special challenge occurred that slowed progress with transcriptomic work. Specifically, In November 2018, shortly after the project began, the Camp Fire began in Butte County. This fire was the deadliest and most destructive fire in California history. The smoke across northern California was so thick that University of California in Davis was fully shut for more than 2 weeks because the air was not safe to breath. The air filtering systems in the building where our thrips colony were located became smoky and we noticed a definite response in the insects. Therefore, we had to discard our initial salivary gland dissections and could not continue until the fire was controlled and the air was free of smoke. Consequently, we are approximately two months behind our timeline for completing salivary gland dissections. As mentioned earlier, the first instars are challenging, but we have a good method that will allow us to complete these dissections. What opportunities for training and professional development has the project provided?UC Davis (Ullman): The postdoctoral scholar and now Assistant Project Scientist on the project, Sulley Ben Mahmoud has learned new techniques for salivary gland dissection, RNA extraction, and transient expression of salivary gland transcripts. In recognition of his work, organizers of the National Entomological Society of America meeting in November have invited him to present at a national symposium. With his promotion to Assistant Project Scientist, he has become more engaged in the work needed to run the laboratory and manage the budget. He attends various seminars in Plant Pathology and Entomology. NC State (Rotenberg): Postdoctoral scholar, Priya Rajarapu, has learned about new state-of-the-art proteomic technologies and analysis tools through interactions with DUKE proteomic core facility staff scientists. She has also been reporting/presenting monthly at Zoom `lab meetings' with collaborating PIs and their postdoc and student on this project. University of Cincinnati (Benoit): Graduate students, Christopher Holmes and Sam Bailey, have learned new bioinformatic techniques to examine RNA-seq data sets. Bailey has also begun to learn specific assays to measure nutrient levels of thrips and other physiological aspects. How have the results been disseminated to communities of interest? Nothing Reported What do you plan to do during the next reporting period to accomplish the goals?Plans at each institution are presented below: UC Davis (Ullman): Overcome challenges to remove SGs from L1 larvae, and conclude dissections Submit RNA samples for RNA-seq library preparation and sequencing Perform experiments to determine plant (tomato) defense response to WFT salivary protein products and determine impact on WFT physiology Perform further cloning of WFT-SG transcripts for transient expression in-planta given results of RNA-seq analysis NC State (Rotenberg: Perform TSWV acquisition experiments to dissect SGs from adult male and female thrips to determine the composition and abundance of proteins in response to virus infection. Optimize ways to effectively dissect larval SG tissues for proteomics Collect saliva from larval and adult thrips and perform proteomic analysis to determine the composition and abundance of secreted proteins. Compare SG proteomic and transcriptomic data to determine overlap to select candidates for effector functional analysis. University of Cincinnati (Benoit): Perform initial RNA-seq studies from sexes and developmental stages (both infected and uninfected). Perform baselines stress tolerance for infected and uninfected thrips. Examine nutritional reserve levels for infected and uninfected thrips. Establish if infection alters thrip behavior such as circadian rhythm changes,

Impacts
What was accomplished under these goals? This section is presented by research institution and Aim. UC Davis (Ullman): General: We have made significant progress on SG dissections, and settled on an appropriate total RNA extractions technique. We have established cloning approaches to transiently express candidate thrips SG transcripts. Meanwhile, transformation of tomato with WFT-SG constructs is on-going. Aim 1. Test the hypothesis that abundance of WFT SG-enriched transcripts vary with development, sex, and TSWV infection status. Dissections of SGs from TSWV-infected and non-infected larvae, and male and female adults are ongoing. We have completed 3-4 biological replicates, consisting of 50 pooled SGs from the aforementioned WFT stages have been completed. Whereas there have been technical challenges removing SG from L1 larvae, mostly due to their relatively small size, we are working to improve the technique to bring the task to completion. Several methods for RNA extraction have been evaluated with midgut tissues and a TRIzol® protocol that includes phenol and phenol:chloroform precipitations to remove DNA and protein contaminants, concluding with Lithium Chloride combined with glycerol precipitation has worked well to yield RNA of the quality and quantity needed for RNA-seq library preparation. We will adapt the TRIzol® technique to extract total RNA from thrips SG. Within the next three months, we expect to submit samples for RNA library preparation and sequencing followed by analysis by Joshua Benoit's laboratory at University of Cincinnati. AIM 3: We will use phenotypic screening to test the hypothesis that WFT salivary proteins from NV- and V-insects may differentially modulate plant defenses against WFT and TSWV. Our preliminary transcriptomic analyses identified a set of salivary proteins of interest. Based on these findings, we have cloned and sequence verified selected western flower thrips (WFT) salivary gland (SG) enriched transcripts into the plant expression vectors: pK2GW7 (donated by Neelima Sinha at UC Davis), pYL254 and pSPDK2483 (donated by Dinesh Kumar at UC Davis). In July, 2019, two pK2GW7:WFT-SG constructs were been submitted to the Plant Transformation Facility, at UC Davis for tomato (Money maker) transformation. We expect to receive the first transformed plants in November 2019. In addition, we are working towards identifying additional potential effectors within candidate SG-enriched transcripts with Agrobacterium-mediated transient expression experiments in-planta (Nicotiana benthamiana). We have tested multiple methods and found great success with a dip method that we favor because no mechanical damage is done to the plant during introduction of the transcripts. We have detected transcript expression via conventional RT-PCR. Currently, we are testing this transient expression method in tomato and, we will analyze protein expression using protein gel electrophoresis and western blotting. Next, we will conduct experiments to determine impact on plant defense genes and WFT physiology. It is necessary to determine protein expression in tomato for the purpose of future WFT leaf-disc bioassays because thrips will not feed on N. benthamiana, but thrive on tomato leaves. We have well established leaf-disc bioassay protocols in the Ullman laboratory. NC State (Rotenberg): Aim 2. Test the hypothesis that composition and abundance of salivary gland enriched proteins vary with development and sex, and that TSWV infection modulates expression of these proteins. We optimized our salivary gland (SG) dissection technique for male and female thrips to generate high yields and high-quality preliminary, proteomic outputs. As planned, The Duke Center for Genomic and Computational Biology, Proteomics and Metabolomics Core Facility performed a `trial run' of two protein samples derived from pools of non-infected female (100) or male (200) SGs using qualitative 1-dimensional ultra-performance liquid chromatography-tandem mass spectrophotometry (1D UPLC-MS/MS) with the appropriate internal standards and embedded quality control pools at each stage of the workflow. Our bioinformatic analyses revealed 1,880 and 2,056 proteins identified from female and male salivary glands, respectively, and combined there were 2,152 non-redundant proteins representing the adult thrips proteome. Approximately 95% of these proteins were assigned provisional functional annotations using the NCBI non-redundant protein database, with top matches to F. occidentalis (genome and predicted protein sequences) as expected. Among these, 53% of the proteins were assigned gene ontology terms in Blast2GO. TSWV acquisition experiments using Datura stramonium are under way to obtain pools of infected and non-infected thrips for quantitative 1D UPLC-MS/MS using the optimize parameters determined by this team, and we are currently optimizing saliva collection from male and female thrips. University of Cincinnati (Benoit): General: We have established specific areas to hold and rear thrips for the proposed physiological studies. Permits are in process to obtain both viral samples and thrips from the Ullmann lab (UC Davis) and Rotenberg lab (NCSU). A graduate student (Bailey) has been recruited to focus solely on this project. Aim 1: Test how TSWV infection status, developmental stage and sex effect expression of WFT SG-enriched genes. We have already identified key salivary genes based on data from the thrip genome project. With this data, we have established bioinformatics pipelines that we will use on future RNA-seq data. This includes the implementation of advanced expressional analyses, including weighted correlation network analysis (WGCNA) to ensure that all analyses are thorough and complete. Aim 3: Test how differentially expressed proteins from non-infected and infected WFT may vary in modulating plant defenses against the insect and the virus it transmits (TSWV) by phenotypic-screening of plants transiently-expressing SG-enriched proteins. We have developed assays to measure lipid, protein, carbohydrate, and glycogen content from infected and uninfected thrips. We have also piloted initial studies on measuring shifts in water content and changes in behavior (activity, circadian rhythms, etc.) that will be examined in both infected and uninfected thrips.

Publications

• Type: Journal Articles Status: Published Year Published: 2018 Citation: Rotenberg, D.* and Whitfield, A. E. Molecular interactions between tospoviruses and thrips vectors. Current Opinion in Virology 2018, 33:191 -197. *corresponding author (doi: /10.1016/j.coviro.2018.11.007)