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