PLANT VIRUS NANOPARTICLE TECHNOLOGY AS PLANT VACCINES

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: COMPLETE
• Funding Source: AFRI COMPETITIVE GRANT
• Reporting Frequency: Annual
• Accession No.: 1028299
• Grant No.: 2022-67012-36698
• Cumulative Award Amt.: $225,000.00
• Proposal No.: 2021-08403
• Project Start Date: Mar 1, 2022
• Project End Date: Jul 31, 2024
• Grant Year: 2022
• Program Code: [A1511]- Agriculture Systems and Technology: Nanotechnology for Agricultural and Food Systems

Recipient Organization
UNIV OF CALIFORNIA-SAN DIEGO
9500 GILMAN DRIVE
LA JOLLA,CA 92093

Performing Department
NanoEngineering

Non Technical Summary
This project utilizes a non-infectious plant virus nanoparticle fordelivery of mRNA into plants, allowing for a temporary change to the behavior of the plants without permanently altering their genetic material. This can allow plants to be temporarily equipped to resist disease, overcome drought conditions, or perform a new function without changing its DNA or creating regulatory hurdles for production. As the human population continues to grow, it is important that we have more techniques to address food security, and mitigating the loss of crops is one of the most challenging issues in this area. Our ultimate goal is to stimulate a common immune response found in a many plants using this technique so we can have a generalized "plant vaccine". In many ways, the principles of this technology are similar to those utilized in the lipid nanoparticle COVID-19 vaccines, but adapted for use in plants. In this work, we will first work to engineer the virus nanoparticles to deliver the appropriate mRNA by breaking them apart, removing their original genetic material, and replacing it with mRNA for the expression of a green fluorescent protein before reassembling the particles. We will then introduce these particles to a culture of cells derived from plants as a proof of concept. The cells that uptake the viruses should begin to glow green as they express the delivered mRNA, and we will use this information to see how efficient the delivery is and how long the cells stay green. After some optimization, we will apply these nanoparticles directly to several plants using abrasion, injection, and passive diffusion to see if their tissues begin to glow green as well. With this information, we can see if any of the nanoparticles need to be improved for higher delivery efficiency. If they do need to be modified, we will add cell-penetrating peptides to their surface which allow the nanoparticles to embed themselves in the cell wall and membrane of the plants for faster uptake. Once we have successfully demonstrated our technology using the green fluorescent protein, we will move on to delivering some transcripts in our nanoparticles that may stimulate an immune response in plants. We will use analytical techniques to see if the expression of different genes, the metabolism, and the proteins in these plant cells have changed due to the delivery of these transcripts. If the results indicate we may have activated an immune response in cell culture, we will advance to whole plant delivery and use the same analytical techniques, imaging, and histology to determine the efficiency of our technology. It is our hope that this technology will enable broad protection against many different pathogens using the highly conserved systemic acquired resistance pathway found in a large number of plants. Specifically, we hope this can be used as an on-demand vaccine for plants when infection is detected somewhere in the field to protect the rest of the crops, which would simultaneously prevent crop losses and improve the economic outlook of farmers in disease-prone areas.

Animal Health Component

50%

Research Effort Categories

Basic

25%

Applied

50%

Developmental

25%

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
215	1499	2020	60%
201	1499	1040	15%
215	1499	1090	15%
206	1499	1030	10%

Knowledge Area
206 - Basic Plant Biology; 215 - Biological Control of Pests Affecting Plants; 201 - Plant Genome, Genetics, and Genetic Mechanisms;

Subject Of Investigation
1499 - Vegetables, general/other;

Field Of Science
1040 - Molecular biology; 1090 - Immunology; 2020 - Engineering; 1030 - Cellular biology;

Keywords

nanoagriculture

plant immunity

viral nanotechnology

Goals / Objectives
This project seeks to create an on-demand "plant vaccine" which will help protect crops when diseased plants are detected nearby. To achieve this, we will use a non-infectious plant virus nanoparticle derived from tobacco mild green mosaic virus. This carrier has been successfully demonstrated in several agricultural applications approved by the USDA and EPA. The virus nanoparticle will be equipped with mRNA to stimulate an immune response in the plants and put them in a self-protective state. Specifically, we hope to stimulate the systemic acquired resistance pathway, which is found across many types of plants and gives long-term and broad-reaching immune protection to the plant. In many ways, the approach is similar to that of the COVID-19 mRNA vaccines, but adapted for plants and their immune systems. If successful, the technology developed in the project will serve as an environmentally friendly and biodegradable nanoparticle that can be directly applied to plants as a pre-exposure prophylactic when disease is detected in the field. This will help to reduce the environmental impact of pesticide application, will mitigate the persistence of unwanted chemicals in the soil and groundwater, and will serve as an effective strategy to protect valuable crops. This will lead to a more sustainable, economically viable, and health-focused approach to crop protection than some of the current methods. The project will also uncover some fundamentals about using protein-based nanocarriers for delivering a temporary change in the cells of plants. This work will also uncover new details about stimulating the systemic acquired resistance pathway and how effective this induction can be in preventing plant disease.Plant viruses are naturally designed to enter plant cells and deliver mRNA, making them an ideal carrier for the immune-stimulating transcripts. By decorating the exterior of these viruses with cell-penetrating peptides, we hope to increase how effective these carriers are at entering the cells and delivering their cargo. To ensure the system works, we will first load the mRNA for a green fluorescent protein into the viral nanoparticles. If successfully delivered, this transcript should direct the cells to produce a green fluorescent signal. The objectives of the project are as follows:1) We will test the system in a liquid culture of plant cells first to see how toxic the viral nanoparticles are when they deliver their cargo. We will use fluorescent microscopy and cell-viability assays to determine the effectiveness and safety of this approach.2) Once confirmed, we will advance to using a variety of whole plants at several stages of development, seeing which tissues express green fluorescence, the timescales to see this fluorescence, and how the health of the plants changes with the treatment.3) Once all the relevant information from green fluorescent expression has been determined, we will introduce candidate sequences of mRNA to turn on the systemic acquired resistance pathway and see which sequences protect the plants from an immune challenge such as introduction of bacteria or fungi.

Project Methods
The strategy of this project is to create a reassembled tobacco mild green mosaic virus (TMGMV) nanocarrier which contains the mRNA for green fluorescent protein (GFP) as a proof of concept, before advancing to candidate mRNA sequences for the stimulation of the systemic acquired resistance (SAR) pathway. We choose to use GFP mRNA as it is a powerful tool to assess the uptake, expression, and relevant biological timescales for delivery of nucleic acids by a rod-shaped virus-like particle (VLP) into plant cells. TMGMV was selected due to its regulatory status, its stability, and its surface chemistry which allows for site-specific chemical conjugation. Once the platform is established and optimized for uptake and expression, we will have created an enabling technology for mRNA delivery into plant cells using a non-toxic nanoparticle. We then will adapt this system for stimulating the systemic acquired resistance response in plant cells to serve as a pre-exposure prophylactic for plants which may be exposed to a variety of pathogens.The efforts we will use to share changes in knowledge with our audience are frequent publications and presentations of our work at scientific conferences and in academic journals. We believe this is the appropriate medium to communicate our findings with the academics and industry representatives who could benefit from our work.The evaluation of this project will be taken at several key junctures. The first step is to make sure we can successfully synthesize TMGMV with mRNA. While this should be trivial, it has yet to be demonstrated explicitly in this virus. We will use standard virus characterization techniques such as size-exclusion chromatography, gel electrophoresis, dynamic light scattering, and mass spectroscopy to confirm the quality of these VLPs. The next evaluation point will be the uptake of these VLPs into the T87 suspension culture. This will be achieved using confocal microscopy, flow cytometry, and commercially available viability kits. If the project is not successful initially, this result can be circumnavigated by advancing directly to the CPP-conjugated VLPs, which have previously demonstrated successful uptake into these cells on different carriers. The next evaluation checkpoint will be to determine the most appropriate uptake route into whole plants, using either vacuum infiltration, abrasion, or passive delivery. This can be achieved using plant tissue imaging, confocal microscopy, viability assays, and analysis of plant morphology. Based on previous results using other carriers, at least one of these techniques should prove to be successful and expression of GFP in the plant tissues should occur. Analogous studies using other carriers have been conducted on all aspects of the project up to this point, so there should be room for optimization to achieve similar results using our system. The most challenging and most rewarding aspect of this project will be the upregulation of SAR using this platform. To demonstrate success in this approach, we will need to utilize techniques such as proteomics, transcriptomics, and metabolomics to see how the cells from several plant tissues are reacting to the mRNA we introduce. We will utilize core facilities at our institution to conduct these analyses. Once the cells have been shown to react in a way that suggests the SAR pathway has been stimulated, we can advance to using whole plants and confirm the same changes there, as well as changes in morphology that may occur. We will also use immunohistology and microtoming to see how the tissues in the delivered area have changed. If these changes seem to indicate a response is occurring, we can work with collaborators to treat and challenge these plants with pathogens and see if the upregulated SAR pathway can function as a pre-exposure prophylactic.

Progress 03/01/22 to 07/31/24

Outputs
Target Audience:chemists, plant biologists, drug delivery scientists, agricultural scientists, chemical engineers, protein scientists, regulatory agencies, tech transfer Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?This project provided the Project Director (PD) with several professional development and training opportunities. In particular, it funded research that led to a number of publications and conference opportunities to network with, present to, and learn from leaders in the field. Additionally, by having consistent funding over the course of the project, the PD was able to have a total of 3.5 years of training with his mentor and become a leader in the lab. He mentored many undergraduate students, masters students, and doctoral students during his time in the lab, learned lab management and inventory, and became extremely well-equipped for faculty interviews. The projects proposed during his faculty interviews were very informed by the work on this project, and he secured a faculty position at Northeastern University after on-site interviews at four R1 chemical engineering programs. How have the results been disseminated to communities of interest?Yes, via publications (see products) and presentations at annual NIFA meetings, including the 2022 and 2024 GRC on Nanoscale Science and Engineering for Food and Agriculture and the NIFA grantee meeting at UT Knoxville in 2023. Additionally, the work was presented at the AIChE annual meeting in 2023 as a oral and poster presentation. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Under the goals of Objective 1, the formulation of nucleic acid-loaded plant virus nanocarriers was further developed. In particular, the preparation of 10 mM MES buffer with pH 6 was found to be the best carrier buffer for agroinfiltration of SNPs containing plasmid. Additionally, we determined that the carrying capacity for plasmid and mRNA loaded SNPs (prepared with 2 mg mL-1 of TMGMV coat protein) was around 700-1500 ng and was dependent on the concentration of magnesium sulfate in solution (up to 100 mM). We further confirmed that using acidified virus to purify away residual viral RNA and isolate coat proteins facilitated the loading of nucleic acids such as plasmid or mRNA into the SNPs. We also determined that the time scales of SNP preparation (<60 s of heating at 98 ºC) did not inactivate RNA or plasmid cargo. These results were also consistent when the TMGMV coat proteins were conjugated with cell penetrating peptides (CPPs) CHKHKHKHK and CRRRRRRRRR using maleimide chemistry and copper-assisted azide-alkyne cycloaddition reactions. Under the goals of Objective 2, N. benthamiana plants were injected with nanocarrier doses equivalent to 10-20 µg of nucleic acid per plant at 4 weeks of growth. Photos post-injection at several concentrations of SNP (with and without CPPs) and virus formulations were taken over the course of 10 days using UV lamps to test for expression of GFP from the delivered plasmids or RNA transcripts. No observable effects for GFP expression even with the conjugation of CPP. We did notice the deactivated virus may have triggered localized immune response in plant, which will be investigated in future studies. SNPs have been identified to be too large and/or incapable of delivering nucleic acids across the plant cell wall after agroinfiltration. However, the preparation and formulation of the materials, the design of plasmid and mRNA, and the strategy/dosing for these types of studies is clearer now. Reassembling the virus around new nucleic acid cargo and testing the rod-shaped nanocarriers is a logical next step in these studies. It will be a fruitful avenue to continue investigating the best version of the plant virus nanocarrier to test the hypothesis of delivering nucleic acids related to the NPR1 pathway. Due to challenges in Objective 2, no additional progress was made for Objective 3. The design of the mutation of NPR1 to express a monomeric protein and directly as a transcriptional activator without external stimulus was the main innovation this year. This will be tested in future studies using the plant virus nanocarrier or other gene delivery platforms for proof of concept.

Publications

• Type: Journal Articles Status: Submitted Year Published: 2024 Citation: A.A. Caparco, I. Gonzalez-Gamboa, S. Chang-Liao, N.F. Steinmetz. Avidin-biotin interactions enable functional loading of nematicides on TMGMV. Submitted August 2024 to Journal of Nanoparticle Research.
• Type: Journal Articles Status: Published Year Published: 2024 Citation: Md. R. Islam, M. Youngblood, H. Kim, I. Gonzalez-Gamboa, A.G. Monroy-Borrego, A.A. Caparco, G.V. Lowry, N.F. Steinmetz, J.P. Giraldo. DNA Delivery by Virus-Like Nanocarriers in Plant Cells. Nano Lett., 2024. DOI 10.1021/acs.nanolett.3c04735.
• Type: Journal Articles Status: Submitted Year Published: 2024 Citation: P. Opdensteinen, A.A. Caparco, N.F. Steinmetz. "Delivery of dsRNA with spherical protein nanoparticles for control of nematodes with RNA silencing." Submitted 2024.

Progress 03/01/23 to 02/29/24

Outputs
Target Audience:chemists, plant biologists, drug delivery scientists, agricultural scientists, chemical engineers, protein scientists, regulatory agencies, tech transfer Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Project Director: Mentorship from Prof. Nicole Steinmetz about direction of project and faculty applicantions applied to 25 tenure track faculty positions and had 6 interviews (still in consideration for 4 positions) Management/mentorship of several undergraduate trainees and new graduate students in the research group Lab management skills Future Faculty Workshop in Soft Materials (Texas A&M) UCSD MRSEC Trainee Leadership Advisory Council Member UAW 5810 Financial Secretary How have the results been disseminated to communities of interest?Through manuscripts, presentations, and training of undergraduate students: Manuscripts and Oral Presentations See research products Poster Presentations A.A. Caparco, N.F. Steinmetz. "Protein- and Virus-Based Materials for Environmental and Agricultural Applications." 2023 AIChE Annual Meeting, Orlando, FL, November 2023. Undergraduate Trainees Sabrina Chang-Liaohas completed 2+ years working under this project Won an internal university research fellowship program Conducted an internship at Kite Pharmaceuticals What do you plan to do during the next reporting period to accomplish the goals?Under the goals outlined in Objective 1, the following tasks will be completed: Prepare CPP-TMGMV for plant and plant cell infiltration experiments Probe the behavior of TMGMV and TMGMV SNPs in crossing the cell wall with and without CPPs Under the goals outlined in Objective 2, the following tasks will be completed: delivery and dosing studies for TRBO-GFP plasmids in SNPs toN. benthamiana fluorescent imaging, viability assays,confocal studies delivery and dosing studies for eGFP dsRNA and mRNA toGFP+N. benthamianafor silencing andGFP-N. benthamianafor expression fluorescent imaging, viability assays, confocal studies Under the goals outlined in Objective 3, the following tasks will be completed: preparation of NPR1 and NPR1 (Cys to Ala) mRNA and loading into TMGMV SNPs SEM, agarose gel electrophoresis, SDS-PAGE delivery and dosing of NPR1and NPR1 (Cys to Ala) in SNPs toN. benthamiana viability assays, confocal studies, plant imaging challenge assay to confirm improved protection from SAR proteomicsand qRT-PCR of treated plants before and after challenge to confirm SAR

Impacts
What was accomplished under these goals? Under the goals outlined in Objective 1, the following tasks were completed: Reaction optimization for cell penetrating peptide (CPP) functionalization on TMGMV azide-alkyne cycloaddition and maleimide-cysteine click reactions were assessed for addition of HK4 and R9 CPPs on TMGMV the resulting nanoparticles were characterized by SDS-PAGE, microscopy, and agarose gel electrophoresis Under the goals outlined in Objective 2, the following tasks were completed: Devleopment of TMGMV Coat protein (RNAse treated) approach for nucleic acid (pJL24 plasmid) loading in spherical nanoparticles (SNPs) Loading capacity was determined using agarose gel electrophoresis of the nucleic acid loaded SNPs Particle formation was confirmed by scanning electron microscopy Delivery experiments plasmid loaded in SNPs to N. benthamianaby agroinfiltration ?Using fluorescent photography for GFP signal, tracked signal over several days No signal was observed with pJL24 For plasmid loading, we have pivoted to TRBO-GFP for signal amplification of GFP post inoculation (particle characterization complete) Preparation of dsRNA for gene silencing experiments eGFP was amplified with T7 sites on each terminus and transcribed into dsRNA dsRNA for gene silencing of GFP+N. benthamianawas loaded into SNPs and the capacity was determined Under the goals of Objective 3, the following tasks were completed: Pathway analysis and gene purchase NPR1 has been identified as the protein of interest for systemic acquired resistance (SAR) activation Design of a Cys to Ala mutant of NPR1 to promote transcriptional activator function (and induce SAR) Both versions of the gene have been ordered

Publications

• Type: Conference Papers and Presentations Status: Published Year Published: 2023 Citation: A.A. Caparco, I. Gonzalez-Gamboa, S. Chang Liao, N.F. Steinmetz. Spherical Nanoparticles from TMGMV for Agricultural Delivery of Small Molecules and Nucleic Acids. 2023 AIChE Annual Meeting, Orlando, FL, November 2023.
• Type: Journal Articles Status: Under Review Year Published: 2024 Citation: 12. I. Gonzalez-Gamboa, A.A. Caparco, J.M. McCaskill, P.F. Velazquez, S. Hays, Z. Jin, J. Jokerst, J.K. Pokorski, N.F. Steinmetz. Inter coat protein molecule loading onto TMGMV. Scientific Reports.

Progress 03/01/22 to 02/28/23

Outputs
Target Audience:chemists, plant biologists, drug delivery scientists, agricultural scientists, chemical engineers, protein scientists, regulatory agencies, tech transfer Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?My reearch mentor and I have had many fruitful discussions about my career within the scope of this project. Specifically, we have discussed my future research program if I get a tenure-track position and how the aims of this work can inform my future career. Additionally, we have completed annual IDPs and progress assessments to determine areas of strength and improvement so that I may be as successful as possible. I have applied to and been accepted to several meetings and workshops, giving me the opportunity to network, present my findings, and be trained for my future career. I also hold leadership positions in a few organizations and have received some certifications. Workshops: NextProf Nexus 2022, Berkeley, CA Future Faculty Workshop in Soft Materials (accepted), 2023, College Station, TX Identify ConvergentNanotechnology Approaches for Precision Delivery ofActive Agents in Plants, 2022,Carnegie Mellon University Outreach and Leadership Positions: Science Talks Coordinator, UCSD MRSEC Trainee Leadership Council (affiliate member) UAW 5810 Financial Secretary and Bargaining Team Representative Certificates: SciCom Scientific Communication Workshop, Fleet Science Center, San Diego, CA How have the results been disseminated to communities of interest?Through manuscripts, presentations, and training of undergraduate and masters students: Manuscripts See products Oral Presentations A.A. Caparco, N.F. Steinmetz. "Plant virus nanoparticle technology for precision agriculture." Nanoscale Science and Engineering for Agriculture and Food Systems Gordon Research Seminar 2022, Manchester, NH, June 2022. A.A Caparco., I. Gonzalez-Gamboa, N.F. Steinmetz. "Thermal transformation of rod-shaped viruses into spherical nanoparticles for precision agriculture and drug delivery." ACS Spring 2022, San Diego, CA, March 2022. Poster Presentations A.A. Caparco, I. Gonzalez-Gamboa, N.F. Steinmetz. "Thermal transition of TMGMV to spherical nanoparticles enables encapsulation of hydrophobic cargo." Physical Virology Gordon Research Conference 2023, Barga, Italy, January 2023. A.A. Caparco, N.F. Steinmetz. "Plant virus nanoparticle technology for precision agriculture." Nanoscale Science and Engineering for Agriculture and Food Systems Gordon Research Conference 2022, Manchester, NH, June 2022. Trainees Sabrina Chang Liao, undergraduate researcher for 1+ year Udhaya Pooranam Venkateswaran, Masters student in NanoEngineer, graduated December 2022 Allison McKenzie, undergraduate researcher in MRSEC REU, summer 2022 Erika Aguilar, undergraduate researcher through ENLACE programe, summer 2022 What do you plan to do during the next reporting period to accomplish the goals?In the coming months, we will have much more of a focus on how the materials behave in the plants with and without cell-penetrating peptides now that materials preparations are completed. We will complete Objective 1 and 2, with a primary focus on Objective 2, within the next few months. To have both moncot and dicot plants within the purview of these studies, we will also grow and develop imaging and microscopy techniques for Arabidopsis. Once we complete Objectives 1 and 2, the results will inform our design for Objective 3, as we will be able to discern if plasmid DNA, mRNA, or siRNA will be the most effective approach for nucleic acid delivery in plants. We will then order and deliver nucleic acids related to the immune response of bothArabidopsisandN. benthamianato fulfill the goals of Objective 3.

Impacts
What was accomplished under these goals? Material Preparation:By decorating the exterior of these viruses with cell-penetrating peptides, we hope to increase how effective these carriers are at entering the cells and delivering their cargo. To ensure the system works, we will first load the mRNA for a green fluorescent protein into the viral nanoparticles. In order to complete any of the aims, a successful approach for material preparation needed to be developed. We have successfully disassembled TMGMV and removed its native RNA to prevent plant infection by our materials. Using thermal transformation, we demonstrated its ability to encapsulate Cyanine 5 and other small molecules (Nano Letters manuscript), as well as its ability to be used in soil-based applications (ACS Ag Sci Tech manuscript). We have introduced and encapsulated plasmid DNA TRBO-GFP into TMGMV-based materials by thermal transformation. The resulting spherical nanoparticles have been characterized by scanning electron microscopy, agarose gel electrophoreis, and SDS-PAGE. We have also successfully conjugated the cell penetrating peptides CHKHKHKHK and CRRRRRRRRR onto TMGMV. These will be used in future studies with these materials. The chemistry required to completed these reactions was optimized in a comprehensive analysis of the reactivity of TMGMV using click chemistry (Chembiochem manuscript). Objective 1:We will test the system in a liquid culture of plant cells first to see how toxic the viral nanoparticles are when they deliver their cargo. We will use fluorescent microscopy and cell-viability assays to determine the effectiveness and safety of this approach. We have ordered and culturedArabidopsisthalianaT87 cells. The culturing techniques have been established and the requisite equipment has been purchased. The work for this aim is on-going. Objective 2:Once confirmed, we will advance to using a variety of whole plants at several stages of development, seeing which tissues express green fluorescence, the timescales to see this fluorescence, and how the health of the plants changes with the treatment. We have grownN. benthamianaas our first plant for this aim. We have developed timelines for infection, imaging, and microscopy to capture fluorescence using native TMV and TMGMV. We have recently ordered plants which natively express GFP and siRNA which can silence GFP expression, which will be assessed within the same framework as other nucleic acids in Objective 2. The work for this aim is on-going.

Publications

• Type: Journal Articles Status: Published Year Published: 2022 Citation: I. Gonz�lez-Gamboa, A. A. Caparco, J. M. McCaskill, N. F. Steinmetz, ChemBioChem 2022, 23, e202200323.
• Type: Journal Articles Status: Accepted Year Published: 2023 Citation: Caparco, Adam ; Gonz�lez Gamboa, Ivonne; Hays, Samuel; Pokorski, Jonathan; Steinmetz, Nicole. "Delivery of nematicides using TMGMV-derived spherical nanoparticles." Nano Letters.
• Type: Journal Articles Status: Accepted Year Published: 2023 Citation: Adam A. Caparco, Udhaya P. Venkateswaran, Ivonne Gonz�lez-Gamboa, Reca Caballero, and Nicole F. Steinmetz. "Plant viral nanocarrier soil mobility as a function of soil type and nanoparticle properties." ACS Ag Sci Tech.