Progress 06/01/24 to 05/31/25

Outputs
Target Audience:Target audiences reached by our efforts during this reporting period include: 1. Participants of the 2024 Annual meeting for Entomology Society of America. 2.Graduate students (6students) at the University of Tennessee, to whom PD serves as a member of their graduate committee. 3. Undergraduate students (48 students) who took the Plant Propagation course in Spring 2025, with PD as one of the co-instructors. 4. Faculty at Univerisotgy of Tennessee through the Plant Ecosystem Resilience in a Changing Environment Community of Scholars (PERCE-CoS) program Changes/Problems:Progress on characterizing the enzymes responsible for four of the five steps in the plant-derived JH III biosynthetic pathway has been slower than anticipated. To date, all tested candidate enzymes for these steps have shown no detectable activity under the current assay conditions. This has presented a challenge and temporarily slowed progress on Objective 2. We will continue to pursue this objective by screening additional candidate genes from the remaining lists and optimizing assay conditions to improve detection sensitivity. Importantly, as a parallel strategy, we have successfully demonstrated that a hybrid JH III pathway--comprising insect genes and the validated C. iria FAMT gene--functions effectively in N. benthamiana through agroinfiltration. This alternative approach provides a viable backup for generating stable transgenic plants and will allow us to continue advancing toward our overall project goals. What opportunities for training and professional development has the project provided?This project provided substantial training and professional development for one Ph.D. student and one postdoctoral researcher. The Ph.D. student and the postdoctoral researcher gained expertise in bioinformatic analysis for candidate gene identification, including the use of functional annotation pipelines and comparative genomics. They also acquired workflows for transcriptome-guided gene discovery and received hands-on training in heterologous expression of enzymes in E. coli followed by biochemical activity assays. These experiences deepened their understanding of the JH III biosynthetic pathway and strengthened their skills in both computational and experimental biology. Furthermore, collaborative work with the Ph.D. student provided the postdoc with mentoring experience, fostering a mutually supportive training environment. Together, these training activities enhanced the participants' competencies in molecular biology, metabolic pathway reconstruction, and interdisciplinary research. How have the results been disseminated to communities of interest?At the Annual meeting of Entomology Society of America held in November 2024, PI presented a poster titled "Biosynthesis of insect juvenile hormone in plants: a convergent evolution" based on the data we have obtained thus far. The poster was well received and led to many stimulating discussions. In May 2025, addition, a manuscript detailing the identification and functional characterization of a key enzyme (FAMT) in the C. iria JH III biosynthetic pathway has been submitted to Nature Communications and is currently under review. The title of the manuscript is "Parallel evolution of juvenile hormone biosynthesis highlights metabolic convergence in plants and insects". What do you plan to do during the next reporting period to accomplish the goals?In the upcoming reporting period, we will continue to work on Objective 2 (functionally characterizing the remaining four key biosynthetic genes involved in the production of JH III in C. iria. and initiate experiments for Objective 3 (transfer the complete biosynthetic pathway into a crop plant and assess transgenic lines for insect resistance). On Objective 2, we will continue to characterize the candidates identified, expand candidate screening and refine assay conditions to improve the likelihood of successful enzyme characterization. On Objective 3, we will initiate engineering of the JH III biosynthetic pathway--using a combination of insect-derived genes and the validated C. iria FAMT gene--into N. benthamiana to generate stable transgenic lines. This serves as a contingency strategy if identification of the full plant-based pathway requires additional time. Successful establishment of a functional hybrid pathway in tobacco will enable us to test one of the our most important hypotheses: engineered production of JH III in plants will provide improved resistance to insect pests.

Impacts
What was accomplished under these goals? During this reporting period, we focused on Objective 2: determining the catalytic functions of enzymes encoded by candidate genes identified in C. iria (from Objective 1) through in vitro enzymatic assays. The complete biosynthetic pathway for JH III from farnesyl diphosphate (FPP) includes five enzymatic steps catalyzed by the following enzymes: 1) Farnesyl diphosphate phosphatase (FPPase) - converts FPP to farnesol; 2) Alcohol dehydrogenase (ADH) - converts farnesol to farnesal; 3) Aldehyde dehydrogenase (ALDH) - converts farnesal to farnesoic acid; 4) Farnesoic acid methyltransferase (FAMT) - converts farnesoic acid to methyl farnesoate; 5) Cytochrome P450 monooxygenase (P450) - converts methyl farnesoate to JH III. We generated a prioritized list of candidate genes for each enzymatic step and initiated in vitro functional assays starting with the top candidates. Each gene was cloned into a bacterial expression vector and heterologously expressed in Escherichia coli to produce recombinant proteins. We have successfully identified and validated the C. iria FAMT gene responsible for the fourth step of the pathway. This enzyme was functionally characterized both in vitro through enzyme assays and in vivo via transient expression in Nicotiana benthamiana. These results confirm its role in catalyzing the conversion of farnesoic acid to methyl farnesoate, and a manuscript describing these findings has been submitted for publication. We also tested 6 FPPase candidates, 3 ADH candidates, 3 ALDH candidates, and 2 P450 candidates. Unfortunately, none of these candidates exhibited detectable activity under the assay conditions tested. We are pursuing to address the challenges. The first strategy is to establish a positive control for each enzyme type to verify the enzyme system we are using is optimized. The second strategy is to identify additional candidates from the remaining lists. In summary, we have made significant progress by identifying and functionally validating the C. iria FAMT enzyme involved in JH III biosynthesis. For the remaining four enzymatic steps, we have cloned and tested several candidates and will continue screening and functional characterization to complete the reconstruction of the JH III biosynthetic pathway in C. iria.

Publications

Progress 06/01/23 to 05/31/24

Outputs
Target Audience:Target audiences reached by our efforts during this reporting period include: Graduate students (5 students) at the University of Tennessee, to whom PD serves as a member of their graduate committee. Undergraduate students (about 50) who took the Plant Propagation course in Spring 2024, with PD as one of the co-instructors. Faculty and students at Tennessee State University through a research summit. Changes/Problems:The process of recruiting a postdoc to work on the project was not smooth. One postdoc was recruited before the project got started. Unexpectedly, the individual had an issue of getting visa in time. As a result, the start date of the postdoc was postponed multiple times. The postdoc finally joined the lab in October 2023. Unfortunately, the postdoc experienced health issue after about two months. A decision was made then to have the postdoc to take unpaid leave to undergo treatments. In January 2024, the postdoc informed PD that he would resign due to continued health issue. So PD had to start new rounds of recruitment. While this did slow down the progress of the project, fortunately, the delay was not significant as PD spentmore FTE on the project than planned. The second postdoc has been recruited and will start in August 2024. What opportunities for training and professional development has the project provided?One PhD student had gained extensive training on genomic studies. In particular, the student has established the entire pipeline for transcriptome analysis. The student also gained deeper understanding about the entire biosynthetic pathway of insect juvenile hormone in insects and their possible counterparts in plants. The first postdoc recruited, who had only a short stint due to health reason, also gained training, especially on the aspect of insect-plant interactions. The postdoc did comprehensive literature review in plant substances that have insect hormonal activities. This may eventually lead to a review article. The interactions between the postdoc and the PhD student were mutually beneficial with the postdoc acquiring mentoring experience. How have the results been disseminated to communities of interest?Due to limited results that have been acquired during this first reporting period, our efforts on this aspect is somehow limited. Nonetheless, the PD took the opportunity of a research summit between PD's home institution, the University of Tennessee, Institute of Agriculture and Tennessee State University to introduce the project to faculty of both institutions. What do you plan to do during the next reporting period to accomplish the goals?From the research perspective, the team will focus on objective 2 to identify all key biosynthetic genes of insect juvenile hormone in C. iria. This will be achieved through biochemical characterization of candidate genes that have been identified through objective 1. Each candidate gene will be cloned using RT-PCR from C. iria into a protein expression vector. The resulting construct will be expressed in E. coli to produce recombinant enzymes. Each recombinant enzyme will be tested for hypothesized activities. With farnesoic acid methyltransferase having been identified, the target enzyme to be identified include farnesyl diphosphate phosphatase, farnesol reductase, farnesal reductase, and methyl farnesoate epoxidase. In addition, we will test known insect genes for insect juvenile hormone biosynthesis in plants. Specifically, we plan to test these genes in tobacco using agroinfiltration. This will provide an backup plan in case the identification of insect juvenile hormone biosynthetic genes in C. iria would take a time longer than planned. Should that happen, the insect genes will enable us to proceed with tomato metabolic engineering in year 3 of this project. With more data for farnesoic acid methyltransferase being acquired, we will prepare and submit a manuscript reporting the identification and characterization of this gene from C. iria. This will serve as both an important product and an important way to disseminate our results. In addition, we plan to disseminate our results at professional meetings.

Impacts
What was accomplished under these goals? Insects are major pests of agricultural crops and cause significant yield losses. It is important to identify new solutions of insect control. The long-term goal of this research is to produce insect-resistant crop plants that will benefit both farmers and consumers. This project is novel in that it aims to produce insect resistance by disrupting insect physiology through production of an insect hormone, insect juvenile hormone, in crop plants. Insect juvenile hormone regulates multiple developmental programs of insects, as such, plant-produced insect juvenile hormone can function as a natural insecticide. While crop plants do not produce insect juvenile hormone, the plant Cyperus iria naturally produce insect juvenile hormone. The goal of this project is to elucidate the biosynthetic pathway leading to insect juvenile hormone in C. iria, transfer the pathway into tomato, and test transgenic tomato for insect resistance.This project has three objectives. In Objective 1, the candidate genes encoding the enzymes that catalyze all five enzymatic steps--from farnesyl diphosphate to JH III--will be identified in a model JH III-producing plant, C. iria, through integrated functional genomics. In Objective 2, catalytic functions of the enzymes encoded by candidate genes will be determined through in vitro enzymatic characterization. In Objective 3, the reconstituted multi-gene JH III biosynthetic pathway will be transformed into tomato plants for constitutive production of JH III, and transgenic tomato plants will be evaluated for performance against selected insect pests. During the reporting period, our accomplishments were achieved mainly for objective 1 and partly for objective 2. For objective 1, we first did comprehensive metabolic profiling of C. iria plants growing at different developmental stages and under different conditions. Through this analysis, we identified nine tissues types with difference in the concentrations of insect juvenile hormone and its precursors. With this metabolic information, the nine tissue types were chosen for the production of nine transcriptomes using RNA-seq. From the nine transcriptome, candidate genes for farnesyl diphosphate phosphatase, farnesol reductase, farnesal reductase, farnesoic acid methyltransferase and methyl farnesoate epoxidase were identified. Next, gene expression-metabolic correlation analysis was performed to narrow down the number of candidate genes for each enzyme. With these results, we just got started to work on Objective 2. For which, each candidate gene will be cloned from C. iria into a protein expression vector and expressed in E. coli to produce recombinant enzymes. Each recombinant enzyme will be tested for hypothesized activities. For this objective, biochemical assays for farnesoic acid methyltransferase gene is largely completed. In brief summary, we have been able to identify candidate pathway genes for the production of insect juvenile hormone in C iria, which paves a solid foundation for Objective 2, through which bona fide pathway genes will be identified.

Publications