Source: NORTH CAROLINA STATE UNIV submitted to NRP
MOLECULAR MECHANISMS OF TRANSMISSION INHIBITION BY A VIRAL GLYCOPROTEIN
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
COMPLETE
Funding Source
AFRI COMPETITIVE GRANT
Reporting Frequency
Annual
Accession No.
1014899
Grant No.
2016-67013-27492
Cumulative Award Amt.
$498,783.79
Proposal No.
2017-09097
Multistate No.
(N/A)
Project Start Date
May 1, 2017
Project End Date
Dec 31, 2021
Grant Year
2018
Program Code
[A1121]- Plant Health and Production and Plant Products: Understanding Plant-Associated Microorganisms
Recipient Organization
NORTH CAROLINA STATE UNIV
(N/A)
RALEIGH,NC 27695
Performing Department
Entomology and Plant Pathology
Non Technical Summary
Tomato spotted wilt virus (TSWV) is considered one of the ten most devastating plant viruses due to the ubiquitous nature of the thrips (insect) vector and the extremely wide host range of the virus. TSWV is distributed worldwide, damaging over 1,000 plant species including important commodities such as tomatoes, peppers, lettuce, peanuts, potatoes and many ornamental plants. TSWV and related tospoviruses are considered emerging diseases because in recent years new species have been described, while the plant host range and geographical limits have expanded for established species. The western flower thrips (WFT) is the most efficient TSWV vector and it is an economically important pest in greenhouses and field crops. The management of thrips pests is difficult because populations of WFT have developed high levels of resistance to many insecticides. Transgenic plants are recognized as promising options for tospovirus management. We propose to exploit and elucidate the underlying mechanism(s) and dynamics of transgenic expression of a TSWV glycoprotein for control of tospovirus spread by WFT. TSWV depends exclusively on its thrips vector for plant-to-plant movement; therefore we propose to disrupt the disease cycle by interfering with the early events of virus transmission by the vector. Disruption of virus entry into the vector will prevent insect transmission or greatly reduce transmission efficiency and thus, stop the transmission cycle. TSWV entry into the thrips vector is mediated by the molecular interaction between the virus protein GN, which serves as a viral attachment protein, and the thrips midgut. Based on our recent finding that TSWV-infected tomato transgenics that express GN inhibited TSWV acquisition and transmission by WFT, we aim to determine the mechanism(s) of reduced acquisition from these transgenic plants, determine the efficacy of these transgenics to prevent spread of other related tospoviruses, and define the region of the GN protein that binds to thrips midguts. The outcome of this proposed work will bring us one step closer to our long-term goal of developing novel, sustainable, and biologically-based strategies for controlling TSWV in agricultural croplands and greenhouses.
Animal Health Component
50%
Research Effort Categories
Basic
50%
Applied
50%
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
21214601101100%
Knowledge Area
212 - Pathogens and Nematodes Affecting Plants;

Subject Of Investigation
1460 - Tomato;

Field Of Science
1101 - Virology;
Goals / Objectives
Defining the molecular determinants of a plant virus-vector interaction enables the development of novel virus control strategies that aim to specifically disrupt the interaction. Tomato spotted wilt virus (TSWV) and related tospoviruses are a threat to U.S. agriculture and food security. TSWV acquisition is mediated by the molecular interaction between the virus membrane glycoprotein GN, which serves as a viral attachment protein, and the thrips midgut. Previously, we found that an exogenously-applied soluble form of GN (GN-S) inhibits TSWV binding, acquisition, and transmission to a plant host. We generated transgenic tomato plants expressing a soluble form of GN with a GFP tag (GN-S::GFP) and found that thrips that fed on these transgenics had significantly lower virus titers and adult transmission efficiencies than thrips fed on TSWV-infected non-transgenic tomato plants. These results demonstrate that an initial reduction in virus infection of the larval insect midgut can result in a significant decrease in virus titer and transmission over the life-span of the vector.The long range goal of our proposed research is to further develop and improve the GN-S transgene approach through integration with other genes or transgenes that confer resistance to TSWV and related tospoviruses to enhance the durability of virus resistance. The aim of this proposal is to gain a deeper understanding of the GNS-transgene mediated inhibition of tospovirus transmission and new knowledge of the vector molecular determinants that underpin the GN-S/thrips interaction. The specific research objectives are to:1) Determine the mechanism of transgenically-expressed GN-S::GFP inhibition of TSWV acquisition and transmission by WFT. We will use a combination of virological, biochemical, microscopy, and vector transmission techniques to test three alternative hypotheses;2) Test GN-S::GFP transgenic tomato plants for the ability to inhibit transmission of related and newly emerging tospoviruses. We will determine if the GN-S::GFP transgenic plants reduce transmission of field isolates of TSWV and related tospovirus species (TCSV and the newly described GRSV/TCSV natural M RNA reassortant virus); and3) Define the domains of GN required for binding to thrips midguts. We will identify the specific region(s) of GN that binds to thrips midguts to mediate viral attachment and entry into midgut cells. We will express different domains of the GN protein and measure binding to thrips guts, and ability to inhibit virus acquisition and transmission.
Project Methods
The biological mechanism of inhibition of WFT larval acquisition of TSWV from GN-S::GFP transgenics remains uncharacterized and is the focus of Obj.1. We have generated three possible hypotheses to investigate the underlying mechanism of inhibition. We plan to conduct experiments to test each of these hypotheses empirically. We will use a combination of virological, biochemical, microscopy, and vector transmission techniques to determine the mechanism of transmission inhibition. We will use our 3rd generation (T3) GN-S::GFP transgenics because these plants produce high levels of recombinant protein and are susceptible to virus infection. Knowledge gained from these investigations will shape the development and deployment of this transgenics-based approach to reduce plant-to-plant spread of TSWV.We will determine if the GN-S::GFP transgenic plants will inhibit acquisition and transmission of field isolates of TSWV and related tospovirus species (TCSV and the newly described GRSV/TCSV natural M RNA reassortant virus). Phylogenetic analysis of the genetic structure of TSWV populations sampled from various regions of the U.S. and Europe supported geographical structure, i.e., TSWV ORF sequences, including the GPs, were more similar within regions than between regions in the U.S. This finding underscores the importance of including multiple genotypes of TSWV in plans for designing tospovirus control strategies. In some tomato growing regions of South America, TCSV and the related virus GRSV are more prevalent than TSWV, posing a serious threat to U.S. agriculture. TCSV and a natural GRSV/TCSV reassortant containing the parental M segment of TCSV were found recently in Florida and are an emerging problem for vegetable production in this region as they confound integrated pest control recommendations. Tomato breeding programs in the U.S. have primarily focused on control of TSWV, and with the introduction of new viruses, growers have limited effective control strategies. A comparison between a North Carolina representative isolate of TSWV to the GRSV/TCSV reassortant revealed average nucleotide and amino acid sequence identities of 76% and 81%, respectively, along the GN/GC region of the M segment. Since TSWV, TCSV, and the GRSV/TCSV reassortant share thrips vector species, we will use our established WFT acquisition and transmission assays to monitor virus titer in individual L1 and adult thrips and determine transmission efficiency from GN-S::GFP-expressing transgenic tomato.The goal of Obj. 3 is to identify the specific region(s) of GN that binds to thrips midguts to mediate viral attachment and entry into midgut cells. The TSWV GN protein sequence contains two domains that may be important in virus attachment to thrips. We will focus our efforts on these two regions because they have been shown to be functionally-important in receptor mediated processes in eukaryotic systems. We will express regions of the protein and construct deletion mutants to determine the region of GN-S that binds midguts. Once we have identified the domain(s) that binds, we will make targeted amino acid mutations to define important amino acid residues involved in the thrips-GN-S interaction. Defining the GN-S domains that bind thrips midguts will enable us to design peptides to interfere with TSWV entry to thrips guts. Our experiments are novel and important because using GN peptides to block thrips acquisition of TSWV in vivo may be a viable method of significantly reducing virus transmission to crop plants. Efforts will be made publically available through publication in scientific journals, communication/outreach with the general public and stakeholders, and these groups will provide feedback that will contribute to our evaluation efforts.

Progress 05/01/17 to 12/31/21

Outputs
Target Audience:Target audiences reached include undergraduate students and the postdoctoral researchers that participated in the research and received training by the PIs. Research findings were presented to scientists, extension faculty, and stakeholders at local, regional, and national meetings. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Findings from this project were used inguest lectures in graduate courses (Molecular Plant Microbe Interactions at NCSU and Clemson University) to describe current research on vector transmission of plant pathogens and in presentations to scientists, extension faculty, and stakeholders. The project has contributed to the training of three graduate students (2 Ph.D. and 1 M.S.), two postdoctoral researchers and four undergraduate researchers. How have the results been disseminated to communities of interest?The research was used to educate the stakeholders in a presentation at the Mid-Atlantic Fruit and Vegetable Convention. Hershey, PA. We also presented a poster on TSWV control to stakeholders at the 2021 Tomato Field Day in Mills River, NC. The project research findings and approach were presented at the 2021 Entomological Society of America and American Society for Virology Annual Meetings. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Define the domains of GNrequired for binding to thrips midguts.We have made significant progress towards understanding the role of TSWV GN in virus acquisition by thrips and identification and characterization of thrips proteins that interact with GN. Using phage-display libraries, we identified four regions of GN that serve as potential thrips-binding domains. A TSWV GN peptide library was synthesized and screened against thrips vectors by feeding. We identified three major regions of the GN protein that bind to thrips guts using this library. A complementary approach used a phage display library to identify 7-mer peptides that bind to the F. occidentalis guts in feeding assays. A single peptide was obtained from two independent biopanning replicates indicating that this peptide was strongly enriched. Comparison with TSWV GN amino acid sequence revealed four residues shared with the binding peptide and GN. In TSWV acquisition inhibition experiments, the thrips-gut binding peptide decreased acquisition efficiency in thrips by 57%. The peptide localized to the thrips anterior region of the midgut, the binding site of TSWV. The strong interaction of this peptide with WFT could help to reveal the TSWV attachment site in GN and may be used to develop antiviral and insecticidal thrips-targeting proteins. High-throughput sequencing of the interacting phages was performed and data analysis is ongoing. TSWV GN-interacting proteins (TIPs) in larval guts and adult salivary glands. ?To extend our search for other possible thrips proteins that interact with TSWV and to capture a wide array of F. occidentalis proteins that interact directly with GN during the TSWV infection cycle over development, a membrane-based yeast two-hybrid assay screen (MbY2H) was performed with TSWV GN. The full-length GN coding region was cloned into the pBT3-SUC bait vector and one amplified, non-normalized F. occidentalis cDNA library was cloned into the pPR3-N prey vector. Our project identified 58 putative TIPs, with 41% shared between whole bodies (Schneweis et al., 2017), larval guts (Han and Rotenberg, 2021) and adult salivary glands (Rajarapu and Rotenberg, manuscript in prep). Enriched putative TIPs in thrips larval guts indicates active and complex interactions between virus and gut proteins during the infection cycle, and most putative TIPs were annotated as membrane proteins, indicating activity of virus-vector proteins on cellular/intracellular membranes. These findings provide a comprehensive profile of thrips proteins that interact with TSWV GN and their expression tissues important in virus acquisition and inoculation. Further characterization of these interactions will enable the discovery of determinants of thrips vector competence.

Publications

  • Type: Journal Articles Status: Published Year Published: 2021 Citation: Teh, H.W., Martin, K.M, Whitfield, A. E. Design and validation of plasmid vectors for characterizing protein-protein interactions in Spodoptera frugiperda insect cells. 2021. PhytoFrontiers. https://doi.org/10.1094/PHYTOFR-09-21-0059-TA
  • Type: Book Chapters Status: Awaiting Publication Year Published: 2022 Citation: Whitfield, A.E., Wang, Y.H., Turechek, W.W., Gottwald, T. ,Schneider, W., Lorenzen, M. and Adkins, S. 2021. Novel Strategies for Management of Arthropod Vectors and Vector-borne Vegetable Diseases in Handbook of Vegetable Crop Diseases. Springer Nature. In Press
  • Type: Book Chapters 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.
  • Type: Journal Articles Status: Published Year Published: 2021 Citation: Han, J., Rotenberg, D. 2021. Integration of transcriptomics and network analysis reveals co-expressed genes in Frankliniella occidentalis larval guts that respond to tomato spotted wilt virus infection. BMC Genomics 22, 810. https://doi.org/10.1186/s12864-021-08100-4
  • Type: Conference Papers and Presentations Status: Published Year Published: 2021 Citation: Jinlong Han, Anna E. Whitfield, and Dorith Rotenberg. Identification of a transcriptionally-responsive, TSWV-interacting protein in Frankliniella occidentalis first instar guts. Entomology Society of America National Meeting, Denver, CO, October 31-November 3, 2021.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2021 Citation: Hao Wei Teh, Kathleen M, Martin, and Anna E. Whitfield. Validation of BiFC/Co-IP plasmid vectors to investigate protein-protein interactions in Sf9 insect cells. Annual Meeting of the American Society for Virology, Virtual, July19 - 23, 2021.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2022 Citation: A.E. Whitfield. Tomato spotted wilt virus and related tospoviruses. Mid-Atlantic Fruit and Vegetable Convention. Hershey, PA, February 1-3, 2022
  • Type: Other Status: Other Year Published: 2021 Citation: Invited Talks: A.E. Whitfield. Turning an enemy into an ally: Plant rhabdoviruses as a biotech tool. Leslie Wanner Keynote Lecture at the Mid Atlantic Plant Molecular Biology Society, 38th Annual Meeting, August 16, 2021.


Progress 05/01/20 to 04/30/21

Outputs
Target Audience:Target audiences reached include undergraduate students and the postdoctoral researchers that participated in the research and received training by the PIs. Research findings were presented to scientists, extension faculty, and stakeholders at local, regional, and national meetings. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Findings from this project were used in lectures and guest lectures in graduate courses (Molecular Plant Microbe Interactions at NCSU) to describe current research on vector transmission of plant pathogens and in presentations to scientists and extension faculty. The project has contributed to the training of three graduate students (2 Ph.D. and 1 M.S.), two postdoctoral researchers and four undergraduate researchers. 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? Nothing Reported

Impacts
What was accomplished under these goals? We characterized the conservation of GN interactions with endoCP-GN homologs in related thrips vector species. We identified endoCP-GN as a thrips protein that binds to TSWV GN (Badillo-Vargas and Chen et al., 2019). The endoCP-GN protein of F. occidentalis is a member of the CPR family of cuticle proteins and in silico predictions place it into the RR-1 group (Badillo-Vargas and Chen et al., 2019). CPR RR-1 proteins are often considered to be associated with flexible cuticles. CPRs are phylogenetically classified by their conserved CHB4 domains (proximal to the C' terminus of the protein), as the N' terminus of the protein is highly variable across insect species (Wilson, 2010). Given the robust, direct interaction documented between F. occidentalis endoCP-GN and TSWV GN (Badillo-Vargas and Chen et al., 2019), we were keen to determine if this sequence and its interaction with GN are conserved in other thrips species. MbY2H assays confirmed that the full-length and N-terminal, variable region of the endoCP-GN ortholog in F. fusca interacted with TSWV GN, indicating a conserved interaction with the viral protein for the two thrips species. In reciprocal experiments, we determined that the GN protein of other orthotospovirus species (tomato chlorotic spot virus, impatiens necrotic spot virus, and groundnut ringspot virus) interacted directly with the two Frankliniella endoCP-GN orthologs. These results provide evidence that the GN/endoCP-GN interaction is conserved among thrips vector species and virus species. Determining the significance and functional role of this virus-vector protein-protein interaction in F. occidentalis and other thrips vector species will be the goal of future projects. We identified differentially-responsive genes and co-expression gene networks in larval thrips infected with TSWV. Transmission of TSWV by adult thrips requires virus acquisition during the larval stages, whereby acquisition efficiency of F. occidentalis larvae declines with larval development. To begin to understand the larval gut response to TSWV infection and accumulation, a genome-assisted (F. occidentalis official gene set v.1 (Rotenberg et al., 2019a); and assembled genome (Rotenberg et al., 2019b), transcriptomic analysis of F. occidentalis gut tissues of first (early L1) and second (early and mid L2) instars was conducted using RNA-Seq to identify differentially-expressed transcripts (DETs) in response to TSWV compared to non-exposed cohorts (Han and Rotenberg, 2021). The larval gut responded in a developmental stage-dependent manner (i.e., little to no overlap in responsive transcript sequences), with the majority of DETs (105 transcripts, 71%) associated with the early L1 stage at a time when virus infection is limited to midgut epithelial cells. Provisional annotations of these DETs inferred roles in bio-energy (protein, lipids, carbohydrates) storage, recycling and transport, insect innate immunity, digestion, detoxification, and cuticle development. Weighted gene correlation network analysis (WGCNA, Langfelder and Horvath, 2008) of the RNAseq normalized read count data revealed multiple clusters (i.e., modules) of co-expressed transcripts in L1, with the L1 DETs distributed across 10 modules. Intramodular membership analysis of the DETs revealed two enriched modules designated 'blue' with 23 members and 'light cyan' with 27 members, together accounting for ~ 48% of the DETs, and all but two of the module members were up-regulated in response to TSWV. The blue module contained proteins known in other insects to be involved in protein, lipid and sugar transport and storage in preparation of quiescent periods, or energy-consuming activities or response to environmental stress, and proteolysis and deglycosylation of proteins in the lysosome (recycling). The light cyan module included several proteins associated with cuticles and membranes, lipid metabolism, and several transcription factors. Our previously identified TSWV-interacting protein (TIP), endoCP- GN (Badillo-Vargas and Chen, et al., 2019), was also ranked as the most tightly linked member (hub gene) representing its cluster of 24 co-expressed gut transcripts in the light cyan module. Our findings represent the first gut tissue transcriptome study described for any thysanopteran species, and provides the basis for functional interrogation of thrips proteins for roles in vector competence and design of innovative means for disrupting virus transmission.

Publications

  • Type: Conference Papers and Presentations Status: Published Year Published: 2021 Citation: Han, J. and Rotenberg, D. Integration of transcriptomics and network analysis reveal co- expressed genes in Frankliniella occidentalis guts that are responsive to tomato spotted wilt virus infection. Virtual Southeastern Branch Meeting, Entomological Society of Entomology, March 29  31, 2021. (JH received 1st prize for student oral presentation)
  • Type: Other Status: Other Year Published: 2021 Citation: Invited Talks: A.E. Whitfield. Vector transmission and biology of plant-infecting bunyaviruses and rhabdoviruses. Whitfield, A.E. University of North Carolina-Chapel Hill, Department of Microbiology & Immunology. February 2, 2021. D. 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.


Progress 05/01/19 to 04/30/20

Outputs
Target Audience:Target audiences reached include undergraduate students and the postdoctoral researchers that participated in the research and received training by the PIs. Research findings were presented to scientists and extension faculty at local, regional, and national meetings and seminars. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Findings from this project were used in lectures and guest lectures in graduate courses (Plant Virology, Vector Transmission of Plant Pathogens, and Molecular Plant Microbe Interactions) to describe current research on vector transmission of plant pathogens and in presentations to scientists and extension faculty. The project has supported the training of two graduate students, two postdoctoral researchers and three undergraduate researchers. How have the results been disseminated to communities of interest?The research was used to educate the general public in a presentation through the American Scientist Pizza Lunch Podcast (May 19, 2020). The project research findings and approach were presented at the 2020 Entomological Society of America Annual Meeting and the 2020 Emerging Plant Disease and Global Food Security Symposium. What do you plan to do during the next reporting period to accomplish the goals?Thrips gut-binding peptides identified in phage-display biopanning experiments will be used in immunolabeling experiments with thrips to i) verify that peptides bind to thrips guts and ii) determine the location of peptide biding in thrips guts. We hypothesize that GN domains that mediate virus attachement to thrips guts will bind to the anterior region of midgut region 1, the site of TSWV entry. Peptides will also be using in TSWV acquisition inhibition experiments. We are also characterizing the thrips gut response to TSWV infection using transcriptomics and proteomics. We will use the -omics resources and the GN-thrips interactome (proteins identified in the MbY2H screen) to bioinformatically integrate these finding and identify key players in TSWV acquisition by thrips. We will validate these interactions using protein-protein interaction assays and functional genomics. Analysis of GN-GFP transgenic tomato lines has identified the best material for experiments examining the mechanism of GN inhibition of acquisition.

Impacts
What was accomplished under these goals? As a direct approach to measure GN binding to thrips guts, a TSWV GN peptide library was synthesized. We screened the peptide library against thrips vectors by feeding and identified three major regions of the GN protein that bind to thrips guts. We also used a random peptide library to identify gut binding peptides. We have four candidate peptides that will be used in additional experiments to validate their ability to bind to thrips guts. These experiments will identify regions of the GN protein that bind to thrips guts. We have collected and characterized two new field isolates of TSWV for screening experiments with transgenic tomato plants. One isolate was collected from fresh-market tomatoes grown in Western NC. This is a traditional strain of TSWV and does not break resistance genes typically used in tomato. The second isolate is from processing tomato grown in Central CA. This virus isolate is termed resistance-breaking because it overcomes resistance to Sw-5, the most common resistance gene used for TSWV resistance in tomato. This virus has a single amino acid change in NSm that enables it to overcome host resistance. We tested the collection of GN-S::GFP plants for gene expression using two methods i) visualizing GFP tomato leaf tissue in 96-well microtiter plates using the Biotek plate reader and ii) PCR using primers designed to target the GN-S and GFP genes. We identified thee lines with high level GN-S::GFP expression that will be used in experiments with TSWV. A membrane-based yeast two-hybrid screen (MbY2H) screen using TSWV GN as the bait and a thrips cDNA library as the prey identified more than 100 candidate genes that bind TSWV GN. Some of the interactors could function as receptors or co-receptors for the attachment and the entry of the TSWV into the insect gut. We are using bioinformatics to compare these interactors with transcripts and proteins found in thrips salivary glands and gut tissues.

Publications

  • Type: Journal Articles Status: Published Year Published: 2020 Citation: New Technologies for Studying Negative-Strand RNA Viruses in Plant and Arthropod Hosts. Thomas L. German, Marce D. Lorenzen, Nathaniel Grubbs, Anna E. Whitfield. 2020. Molecular Plant-Microbe Interactions, 33, 382-393.
  • Type: Journal Articles Status: Published Year Published: 2020 Citation: Diversity and epidemiology of plant rhabdoviruses. Ralf G. Dietzgen, Nicolas E. Bejerman, Michael M. Goodin, Colleen M. Higgins, Ordom B. Huot, Hideki Kondo, Kathleen M. Martin, Anna E. Whitfield. 2020. Virus Research 281, 197942.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2020 Citation: Mining the interactions between viruses and their vectors for new control strategies. Whitfield, A.E., Rotenberg, D., Lorenzen, M., Xavier, C.A.D. Entomological Society of America Annual Meeting. November 11-5, 2020.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Recent progress in the search for the elusive tospovirus receptor in thrips. Entomological Society of America Annual Meeting, St. Louis MO, November 17-20, 2019.
  • Type: Conference Papers and Presentations Status: Other Year Published: 2020 Citation: Han, J., and Rotenberg, D. Transcriptomic response of Frankliniella occidentalis guts to tomato spotted wilt virus infection. Emerging Plant Disease  Global Food Security Cluster Symposium: Outbreaks: Tackling Emerging Plant Diseases That Threaten Food Security, NCSU, Raleigh, NC, Jan 10, 2020.
  • Type: Other Status: Other Year Published: 2020 Citation: Plants Get Virus Infections Too! Tales of a Viral Threat to Food Security. American Scientist Pizza Lunch Podcast. May 19, 2020.


Progress 05/01/18 to 04/30/19

Outputs
Target Audience:Target audiences reached include undergraduate students and the postdoctoral researchers that participated in the research and received training by the PIs. Research findings were presented to scientists and extension faculty at local, regional, and national meetings and seminars. Changes/Problems:The lead PI and co-PI moved research institutions during this project. This move resulted in a significant delay with recruitment of students and postdoctoral researchers. Additionally, renovations of the new lab space were delayed by more than seven months. We have requested a no-cost extension to continue the research project. The availability of diverse TSWV isolates and thrips vectors at our new institution has enabled additional research on the durability and spectrum of resistance provided by transgenic GN plants. We have also made progress on the identification of thrips proteins that interact with the GN protein and this is contributing to our understanding GN-mediated inhibition of transmission. We recruited a new student to work on the project and she begin graduate school in August 2019 and an additional postdoc will join the team in November 2019. What opportunities for training and professional development has the project provided?Findings from this project were used in lectures and guest lectures in four graduate courses to describe current research on vector transmission of plant pathogens and in presentations to scientists and extension faculty. The project has supported the training of two postdoctoral researchers and three undergraduate researchers. How have the results been disseminated to communities of interest?The research here also is used to educate participants in the Dwight D. Eisenhower School for National Security and Resource Strategy Agribusiness Industry Study (2018 and 2019). The project researchfindings and approach were also presented at theGenetic Engineering and Society Center Colloquium Series that is live-streamed and archived on Youtube (50+ views). What do you plan to do during the next reporting period to accomplish the goals?We recruited a graduate student and a postdoctoral researcher to work on objectives 1 and 2. These trainees will pursue the objectives as described in the original proposal. A postdoctoral researcher has been hired to conduct experiments described for objective 3. We are following the described plan of work to achieve the three objectives.

Impacts
What was accomplished under these goals? We have completed our characterization of the intermolecular interactions between TSWV structural proteins using BiFC and Y2H assays. These experiments demonstrated interactions between GN and itself and with the GC protein. As a direct approach to measure GN binding to thrips guts, a TSWV GN peptide library was synthesized. We are currently screening the peptide library against thrips vectors by feeding. We also plan to probe the midgut and salivary glands with the peptides and a random peptide library. These experiments will identify regions of the GN protein that bind to thrips guts. We have made significant progress towards the identification of thrips proteins that interact directly with TSWV proteins. Gel overlay assays revealed six thrips proteins that interact with structural TSWV proteins. Two proteins, endocuticle structural glycoprotein and cyclophilin, were found to be robust interactors with GN. Immunolocalization of the proteins in L1s revealed robust expression in the midgut and salivary glands of F. occidentalis. We also observed colocalization of GN with these two proteins in insect cells. We are conducting experiments to determine the conservation of viral-thrips protein interactions among different virus and thrips species combinations. These findings will advance the development of new control strategies for emerging tospoviruses. Membrane-based yeast two-hybrid screen (MbY2H) screens were conducted and we identified 11 candidate genes that bind TSWV GN. Some could function as receptors or co-receptors for the attachment and the entry of the TSWV into the insect gut. We will validate these interactions using Bimolecular Fluorescence Complementation (BiFC) assays and co-IP. We developed a gateway vector system for conducting these experiments in insect cell lines.

Publications

  • Type: Journal Articles Status: Accepted Year Published: 2019 Citation: Ismael E Badillo-Vargas, Yuting Chen, Kathleen M. Martin, Dorith Rotenberg, Anna E Whitfield. 2019. Discovery of novel thrips vector proteins that bind to the plant bunyavirus, tomato spotted wilt virus. Journal of Virology, In press * Selected as a spotlight article.
  • Type: Other Status: Other Year Published: 2019 Citation: Emerging Plant Disease threatens Food Security and National Security. Dwight D. Eisenhower School for National Security and Resource Strategy 2019 Agribusiness Industry Study. Raleigh, NC, March 14, 2019.
  • Type: Other Status: Published Year Published: 2019 Citation: Exploiting the specificity of virus-vector interactions for new disease control strategies. Genetic Engineering and Society Colloquium, Raleigh, NC, March 26, 2019


Progress 05/01/17 to 04/30/18

Outputs
Target Audience:Target audiences reached include undergraduate students and the postdoctoral researchers that participated in the research and received training by the PIs. Research findings were presented to scientists and extension faculty at local, regional, and national meetings and seminars. Changes/Problems:The lead PI and co-PI moved research institutions during this project. This move has resulted in a significant delay with recruitment of students and postdoctoral researchers. Additionally, renovations of the new lab space were delayed by more than seven months. We have requested a no-cost extension to continue the research project. The availability of diverse TSWV isolates and thrips vectors at our new institution has enabled additional research on the durability and spectrum of resistance provided by transgenic GN plants. We have also made progress on the identification of thrips proteins that interact with the GN protein and this is contributing to our understanding GN-mediated inhibition of transmission. What opportunities for training and professional development has the project provided?Findings from this project were used in three graduate courses to describe current research on vector transmission of plant pathogens and in eleven presentations to scientists and extension faculty. The project has supported the training of two postdoctoral researchers and three undergraduate researchers. 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?We are actively recruiting agraduate student and a researcher to work on objectives 1-3. These trainees will pursue the objectives as described in the original proposal. A postdoctoral researcheralready on staff is conducting research for objective 1 and 3 . We are following the described plan of work to achieve the three objectives.

Impacts
What was accomplished under these goals? Determine the mechanism of transgenically-expressed GN inhibition of TSWV acquisition and transmission by WFT. We are exploring three alternative hypotheses for GN-mediated inhibition of acquisition. We have been characterizing the intermolecular interactions between TSWV structural proteins using BiFC and Y2H assays. We are also analyzing siRNA populations in transgenic plants to determine if the plant immune system (RNAi) is activated in transgenic plants infected with TSWV. These findings will advance the understanding of transgenic GN transmission inhibition and inform future work on the utility of this tospovirus-control strategy. Test GN transgenic tomato plants for the ability to inhibit transmission of related and newly emerging tospoviruses. We will determine if the GN transgenic plants reduce transmission of field isolates of TSWV and closely related tospovirus species. Characterization of emerging tospoviruses in the SE U.S. has been performed by co-PI S. Adkins. Adkins has optimized transmission assays for these emerging tospoviruses and they routinely achieve 30-70% transmission with individual insects. Additionally, the coding sequences of diverse tospovirus glycoproteins have been cloned and sequenced: capsicum chlorosis virus (CaCV), chrysanthemum stem necrosis tospovirus (CSNV), groundnut ringspot virus (GRSV), tomato chlorotic spot virus (TCSV) and impatiens necrotic spot virus (INSV). We have identified candidate receptors for TSWV in thrips and we are conducting experiments to determine the conservation of viral-thrips protein interactions among different virus and thrips species combinations. These findings will advance the development of new control strategies for emerging tospoviruses. Define the domains of GN required for binding to thrips midguts. We will identify the specific region(s) of GN that binds to thrips midguts to mediate viral attachment and entry into midgut cells. We have made significant progress towards the identification of thrips proteins that interact directly with TSWV proteins. Gel overlay assays and a membrane-based yeast two-hybrid screen (MbY2H) screens have identified 109 thrips proteins that interact with the TSWV structural proteins. Thrips protein- GN interactions are being validated in vitro and in vivo. We have two top candidate interactors from the gel overlay assays and the MbY2H interactors have been prioritized for validation. Proteins that are validated to interact with GN in vivo will be used to map interacting domains and transmission inhibition. As a direct approach to measure GN binding to thrips guts, a TSWV GN peptide library was made, which will be used for identifying regions of the GN protein that bind to thrips guts and screening more TSWV interacting proteins (TIPs) from F. occidentalis midguts.

Publications

  • Type: Journal Articles Status: Published Year Published: 2018 Citation: Rotenberg, D. and Whitfield, A.E. 2018. Molecular interactions between tospoviruses and thrips vectors. Current Opinion in Virology 33, 191-197.
  • Type: Journal Articles Status: Published Year Published: 2018 Citation: Ismael E Badillo-Vargas, Yuting Chen, Kathleen M. Martin, Dorith Rotenberg, Anna E Whitfield. 2018. Discovery of novel thrips vector proteins that bind to the plant bunyavirus, tomato spotted wilt virus. bioRxiv 416560; doi: https://doi.org/10.1101/416560