Progress 04/01/23 to 10/23/24

Outputs
Target Audience:Individuals served by the project (research portion) include the PD, undergraduate students, PhD students, early career researchers, and engineering faculty. The undergraduate and PhD students include those from underserved populations such as students from Hispanic-serving institutions and R2 institutions. It is important to target these specific groups as they are the future workforce of agriculture and biotechnology and may not have many other opportunities to be exposed to this kind of research. In addition, disseminatingthe results and broad impactsof this projectengaged many engineering PhD students and faculty.This knowledge sharing is important for creating a community of scientists interested in using nanotechnology to address agricultural and sustainable challenges. Individuals served by the project (outreach portion) include undergraduate students, PhD students, and postdoctoral researchers. These students and early career researchers are comprised of first-generation students and historically excluded individuals including women, Hispanic, and African-American scholars. It is important to target these groups as diversifying science and academia is crucial for expanding the workforce and generating new innovations. In addition, some of these individuals come from communities that are actively involved in agricultural practices and thus can further help to disseminate knowledge to inform the general public. Changes/Problems:The major changeis the early termination of the project at UC Riverside. This change is due to the fact that the Project Director obtained a faculty position at Michigan State University and will continue the project at their new institution with their PhD students. What opportunities for training and professional development has the project provided?During this project, the PDtrained threeUCR undergraduates in wet lab skills, materials characterization, molecular biology, and plant biology. In addition, the PD provided additional training in materials characterization of nanoparticles to two graduate students and servedas a resource on electron microscopy and nanoparticle characterizationfor other graduate students and postdocs in the lab. The PD also provided mentorship and educational resources to first-generation students at UCR as a mentor in the UC Riverside First-Generation Mentorship Program. Several professional development activities were alsocompleted during the reporting period. The PDparticipated in a future faculty workshop hosted by Cornell University and attended the MPMI, AIChE, and Nanoscale Science and Engineering GRS/GRCconferences in summer 2023,fall 2023, and summer 2024, respectively. In addition, the PD visitedseveral universities for faculty interviews and networkedwith faculty and students as part of the iinterview. As President of the RPA, the PDarrangedwriting workshops for postdocs at UCR interested in academic careers, facilitated the inclusion of postdocs as mentors in UCR's Graduate Student Mentorship Program, and established the annual UCR Postdoctoral Excellence awards with the Graduate Division. How have the results been disseminated to communities of interest?The research results have been disseminated to communities of interest through informal and formal presentations. The PD has met with several undergraduate and graduate students through UCR's First-Generation Mentorship Program and discussed the project motivation and results with those students. In addition, the PD has presented the research to many engineering students, staff, and faculty at a range of institutions including R2 and land-grant universities. Complementing the research seminars, the PD had many small-group discussions with the students and faculty on sustainable technologies and integrating engineering and agriculture in research. The PD also presented at the 2024 Nanoscale Science and Engineering GRS/GRC, enabling dissemination of the research results to early career researchers and faculty in the field. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Spray-induced gene silencing (SIGS) is an environmentally friendly alternative method of plant disease control that uses RNAs to target and silence pathogen genes. However, these RNAs are vulnerable to the environment and cannot be delivered to many microbes, limiting widespread use of SIGS. Therefore, it is necessary to develop new and sustainable nanomaterials, such as plant-derived nanovesicles (PDNVs) that can enhance RNA delivery to microbes for improved plant disease control. The use of PDNV-based plant control will benefit growers, agricultural companies, and researchers by expanding the range of techniques and materials possible for controlling devastating microbial diseases. In addition, understanding how naturally-derived nanoparticles interact with different microbesinform new disease control strategies and advance our basic understanding of these pathogens. Through this project, a new class of nanomaterials (PDNVs) were isolated, characterized, and used for RNA-based plant protection against different microbial plant pathogens. It was found that the PDNVs were stable at temperatures rangingfrom -20C to 42C for up to one month, which is valuable when thinking about storage and commercialization. Importantly, using the PDNVs to deliver RNA against plant pathogensextended the protection duration against fungal pathogens on plant leaves and fruits and enabled disease protection against oomycete pathogens that previously could not be well-controlled using RNA. Certaincompositions of PDNVs, liketomato-derived PDNVs,were found to be more effective for SIGS-based control than others, which suggests that there are specific material features of these PDNVs that promote antimicrobial activity and RNA delivery relative to PDNVs derived from other plants. Consequently, the lipidcomposition of the different PDNVs and their small molecule cargo was identified using metabolomics.This information will becriticalfor identifying new antimicrobial compounds and designing better nanomaterials for RNA delivery to microbial pathogens. In addition, these experiments allowed for threeundergraduate students and two graduate studentsto gain valuable expertise in microbiology, working with plants, and in characterizing nanomaterials, which will support them in their future scientific careers. Through this project, the PD also obtaineda faculty position at a major land-grant university and all students who worked on this project had positive graduation outcomes. Overall, the work completed through this projectdeveloped a strong foundation for using PDNVs to improve SIGS against microbial pathogens in order to lower agricultural chemical usage and improve food security.

Publications

• Type: Conference Papers and Presentations Status: Other Year Published: 2024 Citation: "Plant-Derived Nanovesicles for Nucleic Acid Delivery to Microbial Pathogens for Spray Induced Gene Silencing and Genetic Engineering"

Progress 04/01/23 to 03/31/24

Outputs
Target Audience:The target audience reached through the research training provided by this project are the PD and undergraduate students whoare members of underrepresented populations in STEM (female, first-generation, Hispanic, etc.). The audienceof the professional development and leadership training aspects of the projectduring this reporting period are the PD, the postdoctoral community at UC Riverside, and UC Riverside students. These communities consist of members from population groups that are historically underrepresented in STEM at these levels including women, first-generation students or those from low-income backgrounds, Hispanic/Latinx, and scholars with families so it is important to continue efforts to broaden their participation in agriculture-related science. Changes/Problems:An unexpected outcome that is good is that the rate of expenditure is slower than expected sincethe PD has been fortunate to have been quite successful during the faculty candidate interview cycle. As a result, the PD has spent several months preparing their faculty application and interviewing, which has delayed the project progress a little. However, this will not impact the research goals. The other challenge observed that contributed to a slower rate of expenditure was slight difficulty with plant growth conditions experienced at UCR. Plant growth was hampered for a period of a couple months due to issues with the water supply and waiting for a part to be delivered. What opportunities for training and professional development has the project provided?During this project, the PD has trained two UCR undergraduates in wet lab skills, materials characterization, molecular biology, and plant biology. In addition, the PD has provided additional training in materials characterization of nanoparticles to a graduate student and serves as a resource on electron microscopy for other graduate students and postdocs in the lab.There have also been several professional development activities that have been provided during the reporting period. The PD has participated in a future faculty workshop hosted by Cornell University and attended the MPMI and AIChE conferences in summer and fall 2023. In addition, the PD has had the opportunity to visit several universities to present research seminars and engage with faculty and students as part of the faculty candidate interview. As President of the RPA, the PD has assisted in arranging writing workshops for postdocs at UCR interested in academic careers, helped to enable the inclusion of postdocs as mentors in UCR's Graduate Student Mentorship Program, and worked with the Graduate Division to runthe 2nd annual UCR Postdoctoral Excellence awards. How have the results been disseminated to communities of interest?The research results have been disseminated to communities of interest through informal and formal presentations. The PD has met with several undergraduate and graduate students through UCR's First-Generation Mentorship Program and discussed the project motivation and results with those students. In addition, the PD has presented the research to many engineering students, staff, and faculty at a range of institutions including R2 and land-grant universities. Complementing the research seminars, the PD had many small-group discussions with the students and faculty on sustainable technologies and integrating engineering and agriculture in research. What do you plan to do during the next reporting period to accomplish the goals?In the next reporting period, the PD will seek to complete objectives 2 and 3 and to publish a manuscript on objective 1. Specifically, the PD is finishing up the final experiments and writing the manuscript for the use of PDNVs for RNA delivery and plant protection. This will be facilitated using brand-new greenhouse space for growing plants. The PD will also begin understanding and optimizing protein and plasmid loading in the PDNVs to establish new transformation protocols for different bacteria and fungi. This will include using fluorescent protein reporters developed by the Judelson lab as cargo in the PDNVs. Finally, the PD will submit selected PDNV samples for analysis by the UCR Metabolomics and Proteomics cores and begin constructing artificial PDNVs to test specific material and chemical features.

Impacts
What was accomplished under these goals? Spray-induced gene silencing (SIGS) is an environmentally friendly alternative method of plant disease control that uses RNAs to target and silence pathogen genes. However, these RNAs are vulnerable to the environment and cannot be delivered to many microbes, limiting widespread use of SIGS. Therefore, it is necessary to develop new and sustainable nanomaterials, such as plant-derived nanovesicles (PDNVs) that can enhance RNA delivery to microbes for improved plant disease control. The use of PDNV-based plant control will benefit growers, agricultural companies, and researchers by expanding the range of techniques and materials possible for controlling devastating microbial diseases. During this reporting period, a new class of nanomaterials (PDNVs) were isolated, characterized, and used for RNA-based plant protection against fungal and oomycete pathogens. It was found that the PDNVs were stable at various temperatures from -20C to 42C for up to one month and could protect RNA from being degraded. Using the PDNVs to deliver RNA against plant pathogens helped to extend the protection duration against fungal pathogens on plant leaves and fruits and allowed the use of SIGS against oomycete pathogens that previously could not be controlled. It was also found that specific compositions of PDNVs (i.e. lime-derived and tomato-derived PDNVs) were more effective for SIGS-based control than others. This suggests that there are specific material features of these PDNVs that promote antimicrobial activity and RNA delivery relative to PDNVs derived from other plants. Finally, these experiments allowed for two undergraduate students to gain valuable expertise in microbiology, working with plants, and in characterizing nanomaterials, which will support them in their future scientific careers. Overall, the work completed in this reporting period lays the foundation for using PDNVsto improve SIGS against microbial pathogens in order to lower agricultural chemical usage and improve food security.

Publications

• Type: Journal Articles Status: Published Year Published: 2023 Citation: Improving RNA-based crop protection through nanotechnology and insights from cross-kingdom RNA trafficking. Current Opinion in Plant Biology, 102441. https://doi.org/10.1016/j.pbi.2023.102441