Progress 04/15/24 to 04/14/25
Outputs
Target Audience:The primary target audience for this project is the larger scientific community of plant researchers investigating plant natural product biology. To this end, I was an invited seminar speaker to the Purdue University Biochemistry Department to talk about poison ivy urushiol chemical biology. I also presented work from this project to the USDA-NIFA Project Directors meeting in Hawaii. A second target audience for this project are students obtaining training in the scientific methods used in this project. One graduate student and three undergraduate students worked in the lab on this project. They obtained first-hand training and supervision by the PD in these scientific methods and reasoning. Students attend weekly group meetings where they reported about their research and obtained additional mentoring by the PD. A tertiary target audience are landscape workers specializing in poison ivy plant removal. I attended an annual conference for these poison ivy removal landscape workers. Changes/Problems:One year no-cost extension to this award: Based on a slow start to the research caused by a one-year sabbatical leave by the PD, and subsequent delays in recruiting a graduate student to the project, the PD and VT SPO requested a pro forma one year extension to this NIFA award. A revised FACE SHEET for proposal 2022-67013-37047 dated 2/10/2025 validated the approved no cost extension with a new deadline of 4/14/2026. Poison ivy cell-free biochemical assays: The USDA panel summary for the awarded project suggested experiments to measure anacardic acid hydroxylase or cardanol hydroxylase enzyme activity in crude cell-free extracts from poison ivy plant tissue. This was a central focus for one undergraduate researcher from the Fall 2023 to Spring 2024. Suffice it to say that no reproducible enzyme activities were detected. There are manifold reasons why this might be the case (e.g. predicted enzyme activities were too dilute and thus under the limit of detection, suboptimal assay conditions, etc.). In the absence of a positive signal, and the good faith effort to pursue this suggested line of research, we did not pursue this avenue of research past the summer of 2024 in part because the Co-PD supervising this work moved his laboratory to another institution. Relocation of Co-PD to a different university: The Co-PD Dr. Pablo Sobrado pursued an endowed faculty chair in the Biochemistry department at the Missouri Science & Technology (M&ST) University in the Fall of 2024. With permission from USDA NIFA, Virginia Tech Sponsored Programs Office made a sub-contract to Dr. Sobrado to continue his role in this project at M&ST in 2025-2026. The Jelesko lab will transfer cDNA sequences of high priority candidate oxidative enzymes to the Sobrado lab for biochemical analysis. What opportunities for training and professional development has the project provided?Graduate student professional development: The first-year graduate student had formal academic course education as well as laboratory training by the PD. The RNAseq transcriptional profiling objective provided training and experience in RNA isolation and Illumina short read data cleaning. In addition, the graduate student was trained in and developed a high level of competency in command line linux bioinformatics and R-studio analyses of the RNAseq data. Undergraduate student professional development: The PD provided direct mentoring of the three UG researchers in the laboratory techniques. Two of the UG researchers did not have prior experience in these laboratory techniques, and they were actively supervised until the PD was confident of the UG researchers had demonstrated adequate skill in these methods. All three UG researchers will graduate with BS degrees in May 2025. The third UG researcher focused on experiments to detect either an anacardic acid hydroxylase or a cardanol hydroxylase activity in poison ivy crude cell free extracts in the Co-PD's laboratory. PD professional development: The PD received formal university-based faculty training in two computational environments: 1) Linux bash shell scripting, and 2) the R studio environment for statistical analysis. This training strengthened the PD's capabilities in mentoring students in these research skills. How have the results been disseminated to communities of interest?Poster Presentation: Lily Lefave and John Jelesko, 2025, Itching to Understand Gene Candidates for Late Steps in Urushiol Biosynthesis. Translational Plant Science Center Symposium, Inn at Virgina Tech, Blacksburg, VA, February 21, 2025 What do you plan to do during the next reporting period to accomplish the goals?PD Jelesko Lab The Jelesko lab will continue to subclone the 18 candidate transcripts into a T-DNA binary vector and then use Agrobacterium tumefaciens transient expression in Nicotiana benthamiana leaves that have been infiltrated with either Anacardic Acid. The leaves will be assayed for both loss of substrate and appearance of urushiol metabolites. Infiltrated poison ivy transcripts that show substrate specific appearance of urushiol will provide the first validation of an enzyme responsible in the late steps of urushiol biosynthesis. These transcripts/cDNAs will be used to design gene-specific RNAi constructs used to produce poison ivy hairy root lines with suppressed mRNA levels. Transcript-specific RNAi-hairy root lines will be assayed for urushiol levels to further validate gene-to-urushiol metabolite associations (as outlined in the proposal). CoPD Sobrado Lab The Sobrado lab will focus first on candidate poison ivy transcripts that show a gene-to-metabolite associations observed in transient N. benthamiana expression assays performed by the Jelesko lab. These transcripts will be used to generate recombinant enzyme in an appropriate microbial expression system. The recombinant enzyme will be purified as biochemically assayed to establish both enzyme kinetics as well as substrate/product specificity for the recombinant enzyme (as outlined in the proposal).
Impacts
What was accomplished under these goals?
Research Objective 1: During the past year, Research Objective 1 was successfully completed. There are currently 18 poison ivy transcripts that met our initial screening criteria consisting of: 1) show > 3-fold differential expression with a P-value <= 0.05 between drupes and leaves, and 2) transcripts showing a significant positive correlation with differential urushiol accumulation in drupes and leaves. These 18 transcripts encoded enzyme families that are consistent with oxidative hydroxylation of aromatic rings (e.g. cytochrome P450s, oxoglutarate-dependent oxygenases, and flavin-dependent mono-oxygenases). In addition, a diverse panel of 18 poison ivy organs were analyzed for both 1) urushiol accumulation levels in conjunction with 2) RNAseq transcript accumulation levels. These data await coordinated weighted gene network analyses to identify other potential metabolic steps in urushiol biosynthesis. Research Objective 2: We are currently subcloning all high priority 18 transcripts (cDNAs) from poison ivy drupe tissue identified in Research Objective 1. Many of these 18 transcripts encode oxidative enzymes with domains that suggest subcellular localization to the endoplasmic reticulum. Thus, proposed expression of the recombinant enzymes in E. coli would be untenable. Therefore, these 18 subclones will be used for future expression of recombinant enzymes in both Nicotiana benthamiana and yeast to evaluate predicted anacardic acid and/or cardanol hydroxylase enzyme activity of the recombinant enzymes. Preliminary Nicotiana benthamiana experiments with anacardic acid or cardanol substrates infiltrated into N. benthamiana leaves demonstrated that both chemicals were still present/stable 24 hours after leaf infiltration. These results indicated that the poison ivy anacardic acid and cardanol metabolites were stable when infiltrated into N. benthamiana leaves, which is a requirement for future analyses of poison ivy recombinant enzyme activity in N. benthamiana leaves. Both of these alternative recombinant protein expression systems were anticipated in the research proposal in the section "Potential pitfalls and work arounds". Research Objective 3: Two part-time undergraduate researchers (student wage employees) were brought onto the project to generate transgenic poison ivy hairy roots as outlined in the proposal. Results to date indicate a very low rate of successful hairy root production.
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Progress 04/15/23 to 04/14/24
Outputs
Target Audience:Graduate student recruitment. After considerable effort to recruit an external graduate student, I was eventually successful. One student that I actively recruited during Spring 2023 declined the offer to join my lab. In Fall 2023, two 1st year Translational Plant Science Graduate students rotated in my lab, but neither accepted offers to join my lab. In Spring 2024, one undergraduate researcher working my lab was successfully recruited to work on this project beginning in summer 2024 as a thesis Masters student. This was a critical step forward for this project. This student is currently working on Research Objective 1 preparing numerous RNA samples for RNAseq. Undergraduate researchers. During this reporting period I personally trained and supervised six undergraduate (UG) researchers working on several aspects of Research Objective 1. One UG researcher worked during the summer of 2023 in both my lab and the Co-PD Sobrado lab. In Fall 2023, two UG researchers worked 6 hrs/week in both the PD and Co-PD laboratories. In Spring 2024 four UG researchers worked in my lab and one in the Co-PD lab. Changes/Problems:Given the delays due to first a one-year sabbatical leave by the PD in 2022-2023 and the recent successful recruitment of a graduate student in the PD's lab, I intend to submit a pro-forma request for a one-time one-year no-cost extension before the end of this award (currently April 14, 2025). This will extend the project until April 14 2026 (i.e. an additional two years from now). In this scenario, I will submit a request through Viriginia Tech Sponsored Programs Office requesting rebudgeting to achieve successful completion of the three research objectives in the next two years. The changes will not alter the scope of the three original Research Objectives, but rather will reallocate current resources to utilize undergraduate researcher wages as a nimble supplemental means to ensure completion of the original three Research Objectives (in particular Research Objective 3 functional genomics objectives requiring plant transformation, QRT-PCR, and urushiol metabolomic profiling on dozens of transgenic lines) in the remaining two-year period. I have found that undergraduate research teams closely trained and supervised by me are an effective (and resilient) means of meeting research objectives. What opportunities for training and professional development has the project provided?As mentioned above, I provided direct mentoring of all seven UG researchers in the laboratory techniques outlined above. None of the UG researchers had prior experience in these laboratory techniques, and they were actively supervised until the PD was confident of the UG researchers had demonstrated adequate skill in these methods. One of the UG researchers will be working on this project the summer of 2024 (supported by a local SURF funding) and will continue through the 2024-2025 academic year. Two current UG researchers will continue on this project during the fall and spring semesters of the next academic year. Two of the seven UG researchers are graduating in May 2024 with BS degrees, and both have are entering Graduate programs in agricultural disciplines. How have the results been disseminated to communities of interest?
Nothing Reported
What do you plan to do during the next reporting period to accomplish the goals?With the start of the full-time graduate student on this project in May 2024 and the continued work of three of the current UG researchers, I anticipate accelerated progress in Research Objective 1 resulting in the identification of several candidate transcripts encoding posited enzymes mediating late steps in urushiol biosynthesis by June 2024. These transcripts encoding putative late steps in urushiol biosynthesis will initiate Research Objectives 2 and 3 focusing on biochemical and functional genomic approaches to validate the posited late urushiol biosynthetic steps and metabolites. The recombinant enzyme production and enzyme kinetic studies will be done under the supervision of the Co-PD Dr. Sobrado. To date, Dr. Sobrado has not successfully recruited a graduate student to Research Objective 2, but he will do so in Fall 2024. The functional genomics using recombinant RNAi poison ivy hairy root cultures and transient expression of recombinant N. benthamiana leaves and the metabolomics of both will be supervised by the PD.
Impacts
What was accomplished under these goals?
A key accomplishment was the recruitment of a MS graduate student to work on this project full time in May of 2024. This individual is currently an UG researcher working in my lab isolating poison ivy RNA for RNAseq analyses. The following two activities were focused on Research Objective 1 (identifying differentially expressed transcripts associated with urushiol biosynthesis). Preparation of diverse poison ivy tissues suitable for both RNAseq transcriptional profiling and clustering of coordinately expressed urushiol biosynthetic genes. We optimized a low salt CTAB RNA isolation procedure that yields high-quality total RNA. We isolated total RNA from nine node-matched drupes and leaves. Over the past six months a team of UG researchers were trained by the PD to germinate and grow poison ivy seedlings in a variety of physiological conditions. These plants were harvested into specific poison ivy organs in different physiological states. These tissues will be used to isolate RNA for RNAseq analyses using Pearson product-moment correlation coefficient clustering to identify groups of coordinately regulated urushiol biosynthetic genes. The above RNAs will be sent for Illumina sequencing by the end of April 2024. Using cell-free poison ivy extracts to measure biochemical activities of posited late steps in urushiol biosynthesis. This experimentation was a recommendation from the review panel. A UG researcher working on this project in the summer of 2023 produced largely uninterpretable experimental results and could not be trusted (and no longer works in the lab). Since Fall 2023 a biochemistry major UG researcher was recruited and has continued working on this aspect of the project. Closely co-supervised by the PD and Co-PD, this UG researcher is currently optimizing the poison ivy cell-free extraction protocol and performing enzyme assays using these cell free extracts. To date, the NADPH-dependent cardanol or anacardic acid oxidase biochemical assays have not yielded a reproducible substrate-dependent NADPH oxidation activity from the cell free extracts. With that said, there are still many avenues for optimization. This subobjective is not essential for this project, but is a worthwhile endeavor because discovery of a validated cardanol or anacardic acid oxidase enzyme activity would greatly narrow the search for the RNAseq transcriptional profiling candidates.
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Progress 04/15/22 to 04/14/23
Outputs
Target Audience:Nine weeks of supervising two rotation graduate students expressing interest in joining this project. One student in Spring 2022 was unsuitable for the project, and the other rotation graduate student is currently working in the lab until May 12th 2023 at which time they will decide if they wish to join this project for their graduate training and research. Changes/Problems:The beginning of this USDA NIFA award in mid-April 2022 was a much-needed resource, but the particular timing posed a challenge for initiating the project. In 2021 I made the decision that I needed to obtain firsthand training and experience in plant genomics so that I can adequately/responsibly supervise graduate students on research projects using NextGen transcriptomics (such as this project). I began a one-year sabbatical leave in mid-May 2022 (approximately one month after this USDA Award Letter). My original plan was to spend four months learning "3rd Generation DNA" sequencing (i.e. new long and accurate ONT DNA sequencing) at the University of British Columbia by sequencing several Toxicodendron genomes and transcriptomes. I also planned a two-month sabbatical research project in Japan sequencing the Japanese poison ivy genome. This original plan would have had me return to my lab at Virginia Tech by November 2022, so that I could host first-year graduate student rotations in my lab from January to May of 2023 for the purpose of recruiting a graduate student to this project. Unfortunately, serial scientific supply chain delays while at the University of British Columbia necessitated postponing my Japan sabbatical research to mid-January to mid-March of 2023. These delays negatively impacted the timing of recruiting graduate students to this project to March - May. On the other hand, these delays had the positive impacts of generating long-read Toxicodendron gDNA and long read cDNA sequences to generate a high-quality reference genome and a long-read cDNA transcriptome resources in support of Research Objective 1 of this project. The Co-PD on this project Dr. Pablo Sobrado (responsible for Research Objective 2) informed me that he was unable to recruit a graduate student to this project this academic year. Recruitment of a graduate student to this project is his top priority for the 2023 Fall academic term. This did not present hardship for the project, because Research Objective 2 (as described in the proposal) requires gene candidates generated from Research Objective 1 transcriptional profiling. With that said, we will follow the additional suggestions of the review panel to use cell-free protein extracts from drupe tissues to perform biochemical enzyme assays (with different co-factors) to identify which enzyme classes are responsible converting anacardic acid to urushiol. During the summer of 2022, I remotely supervised a former undergraduate researcher to collect a large quantity of drupes for biochemical analyses in the Sobrado lab in support of Research Objective 2. As a point of information, the Virginia Tech Sponsored Research Office set up separate internal accounts for the Jelesko and Sobrado labs proportional to the percent effort on the project. In summary, the initiation of this USDA NIFA award one month before my sabbatical research leave entailing two extended international locations far from Virginia Tech, combined with unanticipated delays that extended the duration of those international activities, negatively impacted the recruitment of graduate students to this project. As a point of information, I did not have any existing graduate students to assign to this project when the project was initiated just prior to the start of my sabbatical research leave. Past and current efforts to recruit graduate students to this project: During the 2022 Spring semester (after notification of recommendation for funding, but prior to award letter issuance), I hosted a first-year graduate student lab rotation in my lab. Based on their performance during the laboratory rotation, that student was deemed poorly-suited to work on this project. In the Summer of 2022, I was contacted by a prospective graduate student expressing interest in doing poison ivy research. In December 2022, I hosted a visit of this prospective graduate student to Virginia Tech to actively recruit them to my research program. This exceptionally qualified prospective student is currently visiting other graduate programs to which they applied. To provide an additional enticement, I was able to offer a one-time University fellowship as an incentive to join my lab. This student will make a final decision by April 15, 2023. In addition, since returning from Japan in Mid-March, I am currently hosting another first-year graduate student lab rotation to recruit them to this project. In summary, I am actively recruiting two potential graduate students to work on this project (assuming both wish to commit to this project, I will only accept one). Dr. Sobrado will recruit a graduate student from the pool of Fall 2023 new graduate students. These delays in the first year of the project have reduced the anticipated expenditure rate during the first year. Since returning to Virginia Tech in mid-March 2023, I am directly supervising a laboratory rotation of a first-year graduate student on this project and making expenditures for lab supplies and contractual services described in the budget justification. Specifically, I am personally working in the lab and training the rotating 1st year graduate student to perform mRNA extractions and RNAseq library preparation in pursuit of Research Objective 1. In conclusion, my pursuit of professional development in plant genomics in the form of a sabbatical research leave resulted in delayed initiation of the project. However, the plant genomic skills and materials (gDNA and cDNA sequences) that I developed during this sabbatical research experience will enable me to more effectively train and closely supervise the graduate students to execute the plant genomics aspects of this and future projects. There are no other significant deviations to this project during the first year period. What opportunities for training and professional development has the project provided?Two first-year graduate students doing six-week laboratory rotations in my lab obtained training in plant molecular biology and/or transcriptomics. How have the results been disseminated to communities of interest?
Nothing Reported
What do you plan to do during the next reporting period to accomplish the goals?The highest priority is to recruit a graduate student to this research project. I currently have two students expressing strong interest and will have final decisions no later than May 15th 2023. Since my current sabbatical research leave goes until June 24th 2023, I will work in my laboratory on Research Objective 1 to perform RNAseq analyses comparing poison ivy drupe (with very high urushiol levels) transcript levels to poison ivy leaf (with low urushiol levels) transcript levels to identify differentially expressed transcripts encoding oxidative enzymes consistent with the last step(s) in urushiol biosynthesis. I will continue to work in the lab training a newly recruited graduate student over the summer to insure rapid progress on Research Objective 1. In the unlikely event neither of the current two graduate student prospects can be recruited this spring, I will hire an undergraduate researcher to work on the project this summer and fall, using savings from the currently budgeted GRA line, until a graduate student is recruited to this project.
Impacts
What was accomplished under these goals?
Research Objectives 2 and 3 are largely predicated on the results from Research Objective 1. As explained in the next section, initiation of Research Objective 1 was greatly delayed . Nevertheless, biological and informatic resources in support of Research Objectives 1 and 2 were developed during year 1 of this project. During the 2022 summer months a large number of developing poison ivy drupes (drupes accumulate very high urushiol levels) were collected as tissues suitable for future biochemical assays to determine which type of enzyme(s) are mediating the conversion of anacardic acid and/or cardanol to urushiol (a recommendation of the panel review). Based on both on other poison ivy research in the lab and other laboratory's experience with de novo transcriptomes generated by short-read Illumina DNA sequencing, our published poison ivy de novo short read transcriptome database likely contains many chimeric predicted-transcripts that are artefacts from the transcript assembly algorithm. This problem is common to all de novo transcriptome assemblies from short read DNA sequencing (i.e. Illumina cDNA sequencing). Therefore, during my current sabbatical research leave (see Changes/Problems section below), I performed Oxford Nanopore Technology (ONT) high accuracy long-read sequencing of poison ivy cDNAs, as well as ONT long-read gDNA sequencing of the Toxicodendron radicans ssp. orientale whole genome. These ongoing sabbatical genomic research efforts will result in more reliable poison ivy transcriptome profiling for differentially expressed genes associated with urushiol biosynthesis (Research Objective 1 of this project).
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