Source: NORTH CAROLINA STATE UNIV submitted to
RISK ASSESSMENT OF NEXT-GENERATION VIRAL VECTORS BASED ON PLANT RHABDOVIRUSES
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
ACTIVE
Funding Source
OTHER GRANTS
Reporting Frequency
Annual
Accession No.
1028931
Grant No.
2022-33522-37745
Cumulative Award Amt.
$500,000.00
Proposal No.
2022-03016
Multistate No.
(N/A)
Project Start Date
Aug 1, 2022
Project End Date
Jul 31, 2026
Grant Year
2022
Program Code
[HX]- Biotechnology Risk Assessment
Recipient Organization
NORTH CAROLINA STATE UNIV
(N/A)
RALEIGH,NC 27695
Performing Department
Entomology and Plant Pathology
Non Technical Summary
Plant rhabdoviruses represent the next generation of viral vectors for delivery of proteins and RNAs to plants and insects. Because of their large carrying capacity, there is significant interest in using rhabdoviruses for genome editing. Rhabdoviruses replicate in their plant hosts and insect vectors, thus creating a complex opportunity for understanding risks associated with using these types of viruses as delivery systems. We propose to use our recently developed infectious clone for maize mosaic virus (MMV) as a model for understanding risks associated with rhabdovirus vectors. We will characterize the environmental risk for vector transmission of genetically modified MMV to agronomic and wild grass species (Family Poaceae). We will study virus recombination of the genetically modified virus to determine the potential for genetic exchange between modified MMV and wildtype (WT) MMV. To reduce risk of virus escape and increase utility in the field we will create reduced-risk viral vectors that are competent for plant infection but are no longer insect transmissible. The project outcomes will provide federal regulatory agencies with important information about the specific host range of MMV and the utility of this specific virus vector. The project team is well equipped to conduct the proposed research with genetically modified viruses and vectors and all work will be conducted in proper containment facilities and with appropriate permits.
Animal Health Component
40%
Research Effort Categories
Basic
60%
Applied
40%
Developmental
0%
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
20615101101100%
Knowledge Area
206 - Basic Plant Biology;

Subject Of Investigation
1510 - Corn;

Field Of Science
1101 - Virology;
Goals / Objectives
The next generation of plant virus vectors used to deliver proteins and RNAs to plants and insects is rapidly developing. In the last five years, advancements in technologies have enabled the creation of infectious virus clones for negative-strand RNA viruses (NSRs) including the plant rhabdoviruses, tospoviruses, and emaraviruses. These viruses replicate in their plant hosts and insect vectors, thus creating a complex opportunity for understanding risks associated with using these types of viruses as delivery systems. The plant rhabdoviruses are of particular interest as vector platforms because they can stably carry large cargo. Recent experiments have shown that plant rhabdoviruses can be used to deliver RNA hairpins, proteins (e.g., GFP, Cas9), and guide RNAs to plants. There is significant interest in using rhabdoviruses for genome editing in plant and insect hosts.We propose to use our recently developed infectious clone for maize mosaic alphanucleorhabdovirus (MMV) as a model for understanding risks associated with negative-strand RNA viruses. MMV is an excellent experimental system for exploring the risks associated with NSR viral vectors. MMV was identified as a preferred viral vector for the DARPA "Insect Allies" program because of its narrow plant host range and exclusive reliance on a single planthopper species (Peregrinus maidis) for transmission. MMV is the first alphanucleorhabdovirus with an infectious clone system that has been developed, and nuclear-replicating rhabdoviruses are ideal as expression vectors because they enable proper processing of mRNAs and export from the nucleus. Expression of reporter genes in MMV has shown strong expression up to 8-weeks post-inoculation in maize and was stable over three cycles of transmission. The findings from this proposal will also have direct application because there is significant interest in using MMV as a crop improvement tool. We will use the MMV infectious clone as a model for negative-strand RNA viruses to carefully assess potential environmental risks of these next-generation virus vectors. Our specific objectives are to:•Examine the environmental risk for vector transmission of GE-MMV to agronomic and wild grass species (Family Poaceae).•Determine the potential for genetic exchange between GE-MMV and wildtype (WT) MMV.•Create reduced-risk viral vectors that are competent for plant infection but are no longer insect transmissible.
Project Methods
For objective 1, we will empirically examine the environmental risk for vector transmission of GE MMV to agronomic and wild grass species (Family Poaceae) in containment. Our research goal is to characterize plant host infection, MMV disease development, virus fitness and dispersion of genetically engineered (GE)-MMV.To address objective 2, we will also use the MMV clone in experiments to determine the potential for genetic exchange between GE-MMV and wildtype (WT) MMV. We will characterize the significance of virus recombination on NSRs.For objective 3, we will use basic knowledge of MMV gene function to develop reduced-risk viruses that are competent for plant infection but are no longer insect transmissible. We will introduce mutations and deletions in the viral gene that is required for binding and fusion with animal cells, thus creating a virus that cannot enter insect cells.

Progress 08/01/23 to 07/31/24

Outputs
Target Audience:Target audiences reached include undergraduate/graduate 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 and Vector Transmission of Plant Pathogens 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 graduate students, postdoctoral researchers, and research associates, andundergraduate researchers. How have the results been disseminated to communities of interest?The research was used to educate stakeholders in a presentation at the NCALS Foundation Board and in meetings with industry representatives. What do you plan to do during the next reporting period to accomplish the goals?Experiments related to objective 1 will be finalized and repeated as described above. For objective 2, recombination of MMV will be investigated in the last year of project funding. A third construct with DsRed and MMV-G placed in separate expression cassettes is under development. For objective 3, a new construct for DsRed and MMV-G expression will be constructed. The two genes will be driven by two different Peregrinus maidis promoters. Generation of transgenic planthoppers will enable us to conduct experiments with G deficient virus in the last year of the project.

Impacts
What was accomplished under these goals? ? Examine the environmental risk for vector transmission of GE-MMV to agronomic and wild grass species (Family Poaceae). Natural acquisition experiments were performed to compare acquisition and transmission rates between P.maidis raised on GE-MMV vs WT-MMV infected maize. A lower rate of viral acquisition was detected in adult insects raised on GE-MMV+GFP infected corn as measured by MMV ELISA on individual insects. Moving single insects to new corn plants from the viral acquisition source resulted in a lower percentage of new plants becoming infected with GE-MMV+GFP than WT-MMV. There was also a noticeable decrease in the development of dispersive/macropterous adults when raised on GE-MMV+GFP infected maize. Overall these results suggest multiple factors for virus dispersal are lowered in genetically engineered (GE) MMV relative to the WT virus. We have obtained and tested germination rates of 17 non-maize, landscape grass species commonly found around maize production farms and not previously reported to be hosts of P. maidis or MMV. We are performing non-choice MMV host tests with groups of four grass species from this list plus maize as a control for each trial. To date, we found that one grass species (also grown as an agronomic crop in parts of the world) showed symptoms of discontinuous streaking, and shortened internode length and tested positive for MMV by ELISA. Based on our single, non-choice host test screens, we designed and will launch before the end of 2024 our mesocosm maize and landscape mixed plant species in large dispersion tents at the NCSU Phytotron Plant Science Building. We will have two tents receiving viruliferous males injected with WT-MMV, and two tents receiving viruliferous males injected with GE-MMV+GFP. Dispersion will be allowed to proceed for 1 week, then tents will be gassed to kill planthoppers and the study will proceed for an additional 2 weeks for virus accumulation and symptom development. Determine the potential for genetic exchange between GE-MMV and wildtype (WT) MMV. Although superinfection exclusion (SIE) has been documented to occur for both, +ssRNA and -ssRNA plant viruses, the effect of SIE on viral recombination rates remains understudied. Because cellular co-infection is a prerequisite for recombination to take place, if SIE can limit cellular co-infection of MMV isolates, we expect a reduction in exchange of genetic information to occur. To address this hypothesis, we first verified whether MMV-mediated SIE can occur in plant and insect hosts. We created MMV-GFP and MMV-RFP clones, and these viruses were demonstrated to be viable in the experimental host Nicotiana benthamiana. MMV-GFP and MMV-RFP were transferred and amplified in maize plants. Insects microinjected with MMV-GFP and MMV-RFP showed GFP and RFP fluorescence 5 days after virion microinjection, respectively. Co-microinjection of both viruses demonstrated the potential of insect co-infection to occur. Transmission assays demonstrated that both viruses are transmissible to maize plants, and maize plants showed typical MMV symptoms after virus transmission. Further observations of GFP and RFP fluorescence and immunoblot analyses confirmed virus infection of maize plants. These results confirmed the viability and transmissibility of MMV isolates labeled with GFP and RFP in insect and plant hosts. These viruses were used to test the potential of SIE to take place in plants and insects. To test whether SIE can occur in plants, we performed a series of transmission assays to co-deliver both viruses into the same maize plant. MMV co-infected plants were successfully obtained. In co-infected plants, one virus always outcompeted the other one, suggesting that SIE is occurring. However, a few leaf regions showed overlapping GFP and RFP fluorescence, indicating that cellular co-infection can still occur. Similar results were observed for transmission experiments using two different methods. Similar results were obtained across two independent experimental replications. Whereas these results suggest that SIE is taking place during MMV infection, it may not be 100% effective in limiting cellular-coinfection and further investigation are in progress to verify the temporal aspect of virus delivery into plants and SIE effectiveness in preventing recombination. We further investigated whether SIE could also occur in insects. Our results demonstrate that SIE also occurs in the insect vector and further experiments are underway to test the effect of SIE in virus transmission and recombination rates. Overall, the results demonstrate that SIE is an active MMV-mediated mechanism affecting virus co-infection in plant and insect hosts and can potentially affect recombination rates. Recombination of MMV will be investigated in the last year of project funding. Create reduced-risk viral vectors that are competent for plant infection but are no longer insect transmissible. Rhabdovirus glycoproteins (G) serve multiple roles during the virus infection cycle, including virus cell entry, exit and virion assembly. For plant rhabdoviruses, whereas glycoprotein is essential for virus acquisition, infection and transmission by the insect vector, it is not essential for virus infection in the plant host. Therefore, G protein can be manipulated to create virus competent for plant infection but no longer transmissible. Here we created maize mosaic virus G protein mutants and evaluated the effect of these mutations in virus infection in the experimental host, Nicotiana benthamiana, and in the insect vector, Peregrinus maidis. Based on prediction of G protein domains and putative biological functions played by them during virus infection in plant and insect hosts, three MMV G mutants were created: (i) deletion of the entire gene encoding G protein, (ii) deletion of the N-terminal region of G protein (ectodomain), and (iii) deletion of C-terminal region of the MMV G protein (cytoplasmic domain). Glycoprotein mutants were engineered using a MMV backbone that expresses a GFP reporter gene inserted in between the genes encoding the movement (3) and matrix (M) proteins. Agroinfiltration of MMV derivatives in Nicotiana benthamiana plants confirmed viability of all three mutants at 12 days after agroinfiltration. GFP fluorescence was clearly observed in infiltrated spots. No visual difference was observed in the number of MMV infected cells between MMV control (full-length G) and G mutants. Immunoblot analyses further confirmed the fluorescence analyses. These results further confirm that G is not essential for MMV to establish cellular infection in plant cells as previously observed for another plant rhabdovirus, Sonchus yellow net virus. Further experiments are in progress to further evaluate the effect of G deletion on systemic MMV infection in maize plants. We also verified whether glycoprotein mutants were able to infect the insect vector, Peregrinus maidis. Glycoprotein is involved in virus entry mediated by receptor, and we hypothesized that by deleting the entire gene or essential domains of G would negatively affect virus entry and consequently infection and transmission by the insect vector. MMV mutants were not able to accumulate in insects. In contrast, GFP expression were observed in 100% of insects microinjected with MMV controls. These results reinforce the essentiality of G protein for MMV infection in insects, which is probably involved in virus entry mediated by receptors. Towards the construction of an insect that is competent to transmit MMV deficient in the G protein, two transient expression constructs were made and tested. The first useda constitutive Peregrinus maidis promoter to express the fluorescent protein DsRed first, followed by the self-cleaving peptide T2A, and then the viral glycoprotein MMV-G. In the second construct, the positions of the DsRed and MMV-G ORFs are swapped. No fluorescence was observed in embryos injected witheither construct.

Publications

  • Type: Journal Articles Status: Published Year Published: 2023 Citation: Kanakala, S., Martin, K.M., Xavier, C.A.D., Tran, H. H., Redinbaugh, M.G. and Whitfield, A.E. 2023. Rescue of the first alphanucleorhabdovirus entirely from cloned complementary DNA: an efficient vector for systemic expression of foreign genes in maize and insect vectors. Molecular Plant Pathology, Molecular Plant Pathology 24 (7), 788-800.
  • Type: Conference Papers and Presentations Status: Other Year Published: 2024 Citation: Whitfield, A.E. Dissecting the molecular interactions between plant viruses and their arthropod vectors. Southeastern Regional Virology Conference, April 12-14, 2024, Emory University, Atlanta GA.
  • Type: Conference Papers and Presentations Status: Other Year Published: 2024 Citation: Whitfield, A.E. Emerging Vector-Borne Plant Diseases at the Intersection of Food Security and Health. Keynote Talk at the 2024 Pandemic Prediction and Prevention Destination Area Symposium. May 14, 2024. Virginia Tech, Blacksburg, VA.
  • Type: Conference Papers and Presentations Status: Other Year Published: 2024 Citation: Whitfield, A.E. Plant Viruses as Friends and Foes. Crops Conference. HudsonAlpha Institute for Biotechnology, June 3-6, 2024. Huntsville AL.
  • Type: Conference Papers and Presentations Status: Other Year Published: 2024 Citation: Whitfield, A.E. and Rotenberg, D. Dissecting the molecular interplay between plant viruses and insect vectors. XX International Plant Protection Congress. Athens, Greece July 1-5, 2024. *Keynote talk for session on Plant Pathogens and Insect vector interactions.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2024 Citation: Marc� Lorenzen, William Klobasa, Yu-Hui Wang, C�sar A. D. Xavier, Brad S. Coates, Dorith Rotenberg, and Anna E. Whitfield. Molecular genetic and genomic tools for Peregrinus maidis, an emerging model for the Hemiptera. Entomological Society of America Annual Meeting, National Harbor, MD, November 5-8, 2023.
  • Type: Conference Papers and Presentations Status: Other Year Published: 2024 Citation: Lorenzen, M.D. (2023) Development of genomic resources and tools for a hemipteran pest of maize. 28th IWGO Conference, Nairobi, Kenya
  • Type: Conference Papers and Presentations Status: Other Year Published: 2024 Citation: Lorenzen, M.D. (2024) Trials and Tribulations of Making Transgenic Hemipterans. Entomological Society of Queensland, Queensland, Australia [VIRTUAL]


Progress 08/01/22 to 07/31/23

Outputs
Target Audience:Target audiences reached include undergraduate/graduate 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?Training: Findings from this project were used in lectures and guest lectures in graduate courses (Molecular Plant Microbe Interactions and Vector Transmission of Plant Pathogens 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 2 graduate students, 1 postdoctoral researcher, and 2 research associates, and 2 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 NCALS Foundation Board and in meetings with industry representatives. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Examine the environmental risk for vector transmission of GE-MMV to agronomic and wild grass species (Family Poaceae). Established a reliable source of inocula for GE-MMV and WT-MMV in maize plants Preliminary vector performance tests in prep for virus transmission studies. Conducted experiments to determine the performance of P. maidis females on one wild grass species, and two agronomic grasses. Nymphs emerged from all three plant species, but did not continue to develop and died on the wild grass species and foxtail millet. On sorghum, nymphs developed into adults. Natural acquisition experiments were performed (P. maidis exposed to GE-MMV and WT-MMV infected maize). Acquisition efficiency was very low. Moving to microinjection for vector/virus dispersion experiments. Microinjection of P. maidis adults with GE-MMV and WT-MMV to determine virus titers and inoculation efficiencies. In progress. Determine the potential for genetic exchange between GE-MMV and wildtype (WT) MMV. MMV-GFP and MMV-RFP were created and then amplified in maize plants using vascular puncture inoculation. Large quantities of virus-infected tissue were generated for virus purification. The purified virus will be used to standardize the amount of inoculum given to insects and plants for virus super-infection exclusion experiments and to study evolution. Additional virus constructs for studying MMV evolution and potential for recombination have been designed and are under development. Create reduced-risk viral vectors that are competent for plant infection but are no longer insect transmissible. Multiple constructs for transient and transgenic expression of MMV proteins to complement viruses with deleted genes have been developed and screened. Verification of protein expression using the transient expression assay has not yet been successful. Additional constructs are being developed and transgenic insect development will proceed.

Publications

  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2023 Citation: Never say never: Lessons learned from negative-strand RNA viruses that infect insects and plants. Department of Plant Pathology Seminar Series, Texas A&M University, March 29, 2023.
  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2023 Citation: Towards defining the viral and vector determinants of tomato spotted wilt virus transmission by thrips. 19e Rencontres Virologie V�g�tale - RVV 2023. Aussois France, January 15-19, 2023.
  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2022 Citation: Creating a novel viral vector for genome editing in maize and sorghum: Bayer Grants4Ag Project. Bayer Crop Science Meeting. Raleigh NC, October 24, 2022.
  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2022 Citation: Plant rhabdoviruses for non-transgenic modification of plants and insects. NCALS Research Foundation Meeting. Raleigh NC. November 2, 2022.
  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2023 Citation: C�sar A. D. Xavier and Anna E. Whitfield. Maize mosaic virus mRNA but not its genome is targeted by the RNA interference pathway in the insect vector Peregrinus maidis. The American Society for Virology Annual Meeting, Athens, GA, June 24-28, 2023.
  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2023 Citation: Hao Wei Teh, C�sar Xavier, Marc� Lorenzen, Dorith Rotenberg, and Anna E. Whitfield. Characterization of a Peregrinus maidis host protein which interacts with a viral glycoprotein. Southern Division American Phytopathological Society Meeting, Durham, NC, February 13-16, 2023.
  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2023 Citation: Marc� Lorenzen, William Klobasa, Anna E. Whitfield and Dorith Rotenberg. Roadblocks and Bulldozers: The Challenge of Making Transgenic Hemipterans. International Plant and Animal Genome Conference, San Diego, CA, January 13-18, 2023
  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2022 Citation: Astri Wayadande, William Klobasa, Cesar Xavier, Marc� Lorenzen, Dorith Rotenberg, and Anna E. Whitfield. Stylet probing activities of transgenic and wild type Peregrinus maidis planthoppers. Entomology Society of America National Meeting, Vancouver, British Columbia, November 13-16, 2022.
  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2022 Citation: Kanakala, S., Xavier, C.A.D., Martin, K.M., and Whitfield, A.E. The first Alphanucleorhabdovirus vector system: a versatile tool for gene expression and study of cross-kingdom virus-host interactions. The Annual American Phytopathological Society Meeting, Pittsburgh, PA, August 6  10, 2022.
  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2022 Citation: Teh, H.W., Barandoc-Alviar, K., Rotenberg, D., Martin, K.M., and Whitfield, A.E. Deciphering the interactions between maize mosaic virus and its insect host, Peregrinus maidis. The American Society for Virology Annual Meeting, Madison, WI, July 16-20, 2022.
  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2022 Citation: Xavier, C. A. D., and Whitfield, A. E. Unveiling the molecular aspects of Peregrinus maidis immune response during maize mosaic virus infection. The American Society for Virology Annual Meeting, Madison, WI, July 16-20, 2022.
  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2022 Citation: Illuminating virus biology in plant and insect hosts: The dedication of the Michael M. Goodin Center for Agricultural Fluorescence Microscopy Experiments and Biological Imaging. August 29, 2022, University of Kentucky, Department of Plant Pathology.
  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2022 Citation: What Are Frontiers in Plant Virology After 40 Years of ASV? Plant Virus Interactions with Insect Vectors. 2022. American Society for Virology Meeting, Madison, WI, July 16-20, 2022.
  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2023 Citation: Anne Lindbergh, William Klobasa, Hao Wei Teh, Marce Lorenzen, Anna Whitfield. Characterizing protein interactions in the maize mosaic virus vector system. Summer Undergraduate Research & Creativity Symposium, NCSU, Raleigh NC, July 27, 2023.