Progress 05/01/23 to 04/30/24
Outputs
Target Audience:During the last reporting period, our research efforts reached diverse audiences, including peer scientists, students, and local agricultural communities. We published a manuscript in The Plant Journal (Impact Factor 7), disseminating our findings to an international audience of plant molecular biologists and geneticists. Our presentations at key scientific events such as the American Society of Plant Biologists (ASPB) conference and the Association of 1890 Research Directors Meeting engaged academic professionals, graduate students, and early-career scientists, broadening the impact of our research on peptide signaling and legume root development. Through guest lectures, course development, and mentoring, we directly influenced future scientists, including undergraduate and graduate students, many of whom belong to underrepresented groups in STEM. Graduate student Shivani Dharam showcased her work on histone modifications at multiple research symposia, further expanding our visibility within the scientific community and promoting student-led research. Additionally, undergraduate students presented their findings at conferences that emphasize minority contributions to research, potentially inspiring greater participation in plant biology. Our outreach extended to the local agricultural community at the TSU Small Farm Expo, where PI Roy, and student researcher Shania Dean-Motley engaged minority and smallholder farmers, as well as policymakers, to discuss the real-world applications of our legume genomics research in reducing dependence on nitrogen fertilizers. These engagements fostered connections with a wide range of stakeholders, from academic experts to community growers, reinforcing the societal and scientific relevance of our work. Finally, through the laboratory website legumegenetics.org and social media both individual and through the College of Agriculture's initiaves we promoted the work performed by all project participants. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?GRADUATE STUDENT TRAINING 1. Miss Divya Jain Doctoral student mentored in the lab of lead PI Roy is working on objective 1. Learned how to sterilize seeds, grow Medicago truncatula seedlings using the filter paper sandwich method, treat roots with peptides, and extract DNA using the Qiagen plant mini kit following strict guidelines for whole-genome bisulfite sequencing. Gained experience in preparing DNA samples for bisulfite conversion, fragmenting DNA (200-400 bp), and ensuring quality control using Qubit, real-time PCR, and a bioanalyzer to check library size and distribution before sequencing. Learned to identify DMRs using DSS software, leveraging statistical tests based on spatial correlation, read depth, and variance across biological replicates or pseudo-replicates. Developed skills in statistical interpretation, such as calculating fold change and p-values, to determine significant methylation changes at specific sites. Developed leadership skills and mentored one undergraduate student and four high school students. Gained science communication skills and presented posters and talks at international and National conferences. 2. Miss Shivani Dharam Masters student mentored in the lab of lead PI Roy is working partially on objective 2. Learned to mine databases (MtSSPdb, NCBI CDDB) to identify histone-modifying genes, filter by conserved domains, and prepare FASTA sequences for domain-specific searches. Gained experience in analyzing RNA-seq data, calculating fold changes, log2 fold changes, and performing T-tests to identify significant expression changes during nodulation and peptide treatments. Conducted sequence alignments using MUSCLE and constructed phylogenetic trees using MEGA11 with maximum likelihood methods and bootstrapping for evolutionary insights. Designed CRISPR guide RNAs with CRISPR-P v2.0, optimizing for GC content, off-target minimization, and proximity to PAM sites. Mastered Golden Gate cloning techniques to assemble gRNA sequences into vectors, using type IIS restriction enzymes for precise ligation. Performed PCR amplification, gel electrophoresis, and gel purification to isolate target DNA fragments. Conducted bacterial transformations, plasmid minipreps, and colony PCR to confirm successful insertion of target sequences. Received training in science communication and presented reearch at the Tennessee State University Research symposium (received third prize) and the ARD Symposium in Nashville. UNDERGRADUATE STUDENT TRAINING In addition to Miss Kyla Hughes and Miss Janae Terrell who stayed on the PI and co-PI's laboratories for another year, we have trained Miss Shania Dean-Motley and Miss Aminah Harding-Jones. We anticipate training at least two more undergraduate students over the next year. All students presented their research as posters at the ARD symposium and networked with scientific peers from 1890 schools across the country. 1. Kyla Hughes is an exceptional student specializing in Biotechnology. Under the guidance of graduate student Divya Jain and Dr. Sonali Roy (Lead PI), she has pursued a research project focused on understanding the role of the MtCAPE16 peptide in root development and its potential to enhance plant growth. During the academic year, Kyla collaborated with Divya Jain to identify mutations in MtCAPE16 and two related genes, MtCAPE17 and MtCAPE19, utilizing the CRISPR-Cas9 gene-editing system in M. truncatula. Her project has included the following key tasks: Amplification of target regions within all three genes from 8-10 independent transgenic lines using polymerase chain reaction (PCR). Determination of nucleotide sequences using Sanger sequencing. Analysis of wild-type (unedited) gene sequences retrieved from the National Center for Biotechnology Information (NCBI) to identify open reading frames in both wild-type and mutant sequences. Classification and documentation of sequence variations, including insertions (additional nucleotides) and deletions (missing nucleotides). In January 2024, Kyla was selected to participate in the MIT Quantitative Workshop held in Cambridge, MA. This intensive, week-long program introduces undergraduate students to quantitative data analysis methods and programming languages essential for biological and neuroscience research. 2. Shania Dean-Motley is conducting research under the mentorship of lead PI, Dr. Sonali Roy (Research Professor). Her project utilizes a CRISPR-Cas9 reverse genetics approach to investigate the role of the MtBAK1 gene in root nodule symbiosis in M. truncatula. The study targets all six MtBAK1 orthologs (closely related gene copies) to comprehensively understand the gene's function. Key components of her research are as follows: Designing three specific guide RNAs for each of the six MtBAK1 orthologs. Cloning each guide RNA into the pDIRECT23C vector backbone using Golden Gate assembly. Confirming the sequences of the cloned guide RNAs through Sanger sequencing. 3. Aminah Harding Jones Major: Agricultural Sciences with a Biotechnology Concentration (Freshman) Mentored in the lab of PI, Dr. Sonali Roy (Research Professor), Aminah's research explores the role of BAK1 co-receptor proteins in legume-rhizobia symbiosis using CRISPR-Cas9 genome editing. The project involves a TNt1 mutant collection for comprehensive genetic studies. Key activities include: Employing CRISPR-Cas9 to edit all six MtBAK1 gene orthologs in M. truncatula to study their individual roles in symbiosis. Introducing random genomic mutations through TNt1 mutagenesis. Identifying mutants with disrupted receptor genes, particularly MtBAK1-related co-receptors. Screening for mutants with impaired symbiotic relationships with rhizobia bacteria to determine essential receptors for symbiosis. 4. Janae Terrell Major: Agricultural Sciences with a Biotechnology Concentration (Junior) Under the mentorship of Peter Prestwich (Research Associate) and Dr. Ali Taheri (Associate Professor, Co-PI), Janae's research focuses on the development of soybean-specific CRISPR vectors to enhance gene-editing accuracy. Her project includes the following objectives: Developing CRISPR vectors that incorporate soybean-specific promoters and reporter genes. Designing guide RNAs targeting specific genes in soybeans. Constructing CRISPR vectors using advanced cloning methods. Transforming Agrobacterium tumefaciens with the constructed vectors for plant transformation. Infecting soybean explants with Agrobacterium carrying the CRISPR vectors. Culturing explants to generate hairy roots and regenerating transgenic soybean plants. Verifying gene edits in transgenic plants using molecular biology assays. Assessing the effects of gene edits and confirming the activity of the reporter gene to distinguish transgenic from non-transgenic plants. Refining and optimizing the CRISPR vectors based on research findings. HIGH SCHOOL STUDENT TRAINING PI Roy trained four high school students who participated in the College of Agriculture's Summer Apprenticeship program in June 2023 which acts as one of the most effective tools to recruit African American students to the undergraduate degree program. Four of the forty student cohort chose to work in PI Roy's lab and co-PI Zhou's lab and learn more about the plant 'superpowers' CRISPR can help develop. Students were trained in DNA extraction from CRISPR mutated Medicago leaves, PCR, gel electrophoresis, DNA sequence analysis using NCBI BLAST and Clustal Omega. Students jointly presented a seminar of their research and training achievements at the end of 3 weeks to their peers and family. These students were Joshua Myers (New Manchester High School, Douglasville, GA), Kamola Agzamova (Hume-Fogg High School, Nashville, TN), Chris McKay (Ridgeway High, Memphis, TN) and Anna Qian (Hume-Fogg High, Nashville, TN). How have the results been disseminated to communities of interest?(1) Peer Scientists and students We have published one manuscript over the last year in the Society Journal The Plant Journal (IF 7). We hired and are mentoring one graduate student Miss Shivani Dharam on this project who is working towards a Masters in Agricultural Sciences with a focus in Biotechnology. PI Roy successfully taught a new course on gene editing AGSC4630/AGSC5630 to two undergraduate students and seven graduate students. Co-PI Taheri guest lectured during one class to engage students and talk about delivery of CRISPR constructs into plants. Sonali Roy Oranized two sessions with international speakers at the International Society of Molecular Plant Microbe Interactions on (1) Peptides in plant microbe interactions (2) Mentoring undergraduate student research in plant microbe interactions. Included a graduate and undergraduate student at the session. Invited Speaker: American Society of Plant Biologists, Georgia - Concurrent Session Speaker "Genes to Communities: Uncovering the Impact of GOLVEN10 on Root Nodule Symbiosis through GWAS" Savannah, GA, July 2023 Session Speaker: Association of 1890 Research Directors Annual Meeting, Nashville, TN - "Uncovering the Impact of peptides on Root Nodule Symbiosis and Legume Root architecture" Nashville, TN, April 2023 Invited Speaker: University of Nebraska, Plant Science Departmental Speaker (Virtual) "Peptides in Nodulation and Nitrogen Acquisition" Nashville, TN, June 2023 Invited Speaker: University of Nebraska, Plant Science Departmental Speaker "Peptides in Nodulation and Nitrogen Acquisition" Nashville, TN, April 2023 Invited Panelist: American Society of Plant Biologists, Georgia: Networking workshop - "How to network for a job in Academia" Savannah, GA, July 2023 College wide Speaker TSU, Professional Development Workshop Series for Faculty: High Impact Practices for increasing Undergraduate participation in STEM - "Research with Faculty". April 2024 PI Roy was invited by the University to present her high impact practices on 'Research with Faculty' to peers during the TSU Professional Development Workshop. She talked about practices that help engage undergraduate students in meaningful research within the lab. Divya Jain Joint research talk with Miss Kyla Hughes, Investigating Small Signaling Peptides Involved in Nitrogen Acquisition and Nodulation, International Society for Molecular Plant-Microbe Interactions Congress (2023). Flash talk, CAPE Peptides are involved in Nitrogen Acquisition and Nodulation in Medicago truncatula, North American Symbiotic Nitrogen Fixation Conference, University of Wisconsin (2022). Oral talk, Tennessee Academy of Science, Annual Meeting, Tennessee State University (2022). Shivani Dharam (Graduate Student, Woman) Oral Presentation on research understanding the role of peptides on histone modifications at the 46th Annual University-Wide Research Symposium, Tennessee State University, Nashville, February, 2024. Poster Presentation, Showcased research work regarding Characterization of peptide responsive histone modification genes at Association for 1890's Research Directors Research Symposium, Nashville, Tennessee 2024. Shania Dean-Motley - Presented a poster at the Association of 1890 Research Director Annual Meeting in Nashville, TN (April 2024) Aminah Harding-Jones - Presented a poster at the Association of 1890 Research Director Annual Meeting in Nashville, TN (April 2024) (2) Growers Small farm Expo - Dr. Parmar, PI Roy and undergraduate student Miss Shania Dean-Motley spoke to local farmers, legislatives, and funders at the TSU Small Farm Expo. They explained how their research in fundamental legume genomics could help farmers by identifying bio stimulants that would reduce agricultural dependance on expensive nitrogenous fertilizers. What do you plan to do during the next reporting period to accomplish the goals?During the next reporting this project will (1) In terms of student training and workforce building we willgraduate one doctoral student Miss Divya Jain, and one Master student Ms. Divya Jain.We will alsohire one new student studying towards a doctoral degree. We will train at least two more undergraduate student and four high school students. (2) Since objective 1 and 3 are close to completion, we will focus on completing all tasks associated with objective two. We have already initiated stable transformation of CRISPR-cas mediated Acetyltransferase mutants, we will also generate stable transforation in other three genes. We will complement this with additional molecular biological tasks such as promoter GUS cloning and overexpression in hairy roots. (3) We will finish a manuscript publishing findings we have already generated. This will train a student in writing and scientific publishing as well.
Impacts
What was accomplished under these goals?
Objective 1: Catalogue changes to the status of the nodule methylome upon peptide treatment Objective 1 has been completed. The generated data is being analyzed and will be published in 2025. We treated Medicago truncatula seedling roots with peptides GOLVEN10, CEP1, and CAPE16, both with and without rhizobia, extracted DNA, and conducted Whole Genome Bisulfite Sequencing (WGBS). Below is a summary of the methodology: 1. Genomic DNA Extraction: M. truncatula A-17 seeds were grown on B&D media and treated with peptides for 24 hours. Roots were harvested, frozen at -80°C, and ground for DNA extraction using the Qiagen plant mini kit, followed by sequencing at Novogene. 2. Library Construction and Sequencing: DNA was fragmented (200-400 bp), bisulfite-treated, and prepared into libraries for Illumina sequencing after adapter ligation and PCR amplification. Library quality was assessed using Qubit, real-time PCR, and a bioanalyzer. 3. Data Preprocessing and Mapping: Reads were quality-checked using FastQC and trimmed using Trimmomatic. Clean reads were aligned to the bisulfite-converted genome using Bismark, and duplicates were removed. The sodium bisulfite non-conversion rate was calculated using lambda DNA controls. Methylation levels were visualized using IGVbrowser. 4. Differentially Methylated Region (DMR) Analysis: DMRs were identified using DSS software, which utilized spatial correlation and pseudo-replicates for statistical rigor. Genes associated with DMRs were defined based on overlaps with gene body or promoter regions. Objective 2: Characterize four core chromatin remodeling enzymes targeted by peptide signaling during nodulation Progress has been made toward this objective including identification of genes to be edited and cloning of guide RNAs for an Acetyltransferase. To compile a comprehensive list of histone-modifying enzymes in Medicago truncatula, we employed two approaches: mining the Medicago Small Signaling Peptide Database (MtSSPdb) and cross-referencing publicly available genome data. An initial search yielded 592 genes, from which 271 remained after filtering for relevant histone-modifying genes. A domain analysis using the NCBI Conserved Domain tool further refined this list to 245 genes categorized as histone methyltransferases, acetyltransferases, demethyltransferases, or deacetylases. A final list of 238 genes was compiled after eliminating redundancies and incorporating updates from version 5.0 of the Medicago genome. Expression Analysis: We assessed gene expression of 238 histone-modifying genes during nodule development and peptide treatments using RNA-seq data. Genes were analyzed for fold change across nodulation stages (0, 4, 10, and 14 days post-inoculation) and following peptide treatments (GLV10, IAA, PIP1, and PIP7). Genes with a log2 fold change > 0.58 and p-value < 0.05 were considered significant. A total of 17 candidate genes showed notable expression during nodulation or peptide application, categorized as: Nodulation-induced, peptide-induced: 4 genes Nodulation-induced, peptide-repressed: 4 genes Nodulation-repressed categories were excluded due to lack of relevant phenotypic data. Guide RNAs (gRNAs) were designed using CRISPR-P v2.0, targeting candidate genes with optimal GC content (35-60%) and minimal off-target effects. Golden Gate cloning was performed to assemble gRNA sequences into the pDIRECT23C vector using type IIS restriction enzymes. Colony PCR verified the constructs, but initial Sanger sequencing showed incomplete assembly. Further rounds of Golden Gate assembly and plasmid isolations led to improved constructs. This objective has advanced our understanding of chromatin remodeling genes and their regulation by peptide signaling during nodulation. A second graduate student has been selected and an offer made. She will be joining in August 2024 to work towards goals involved in this objective. Objective 3: Train graduate and undergraduate students in advanced biotechnological tools The project has trained two graduate and four undergraduate students, with additional high school apprentices. 1. Graduate Students: Divya Jain (Ph.D.): Leading Objective 1 research on peptide-induced DNA methylation changes. Shivani Dharam (M.S.): Supporting chromatin remodeling research (Objective 2). 2. Undergraduate Students: Kyla Hughes: Research on MtCAPE16 peptide's role in root development and CRISPR-Cas9 gene editing in M. truncatula. Key tasks: PCR amplification, Sanger sequencing, and mutation analysis. She attended the MIT Quantitative Workshop for advanced data analysis training. Shania Dean-Motley: Investigating the MtBAK1 gene's role in root symbiosis using CRISPR-Cas9, targeting six gene orthologs with guide RNA design, cloning, and sequencing. Aminah Harding Jones: Exploring BAK1 co-receptor proteins in legume-rhizobia symbiosis using TNt1 mutagenesis and CRISPR-Cas9 to identify mutants with disrupted receptor genes and their symbiotic interactions. Janae Terrell: Developing soybean-specific CRISPR vectors with tailored promoters and reporter genes for precision editing. Key tasks include vector construction, Agrobacterium transformation, hairy root culture, and gene edit validation. High School Apprenticeship Program: In June 2023, four students (Joshua Myers, Kamola Agzamova, Chris McKay, and Anna Qian) participated in the College of Agriculture's Summer Apprenticeship program. They learned DNA extraction, PCR, gel electrophoresis, and sequence analysis. The program culminated in a research presentation to peers and family. Classroom Teaching: PI Dr. Roy developed and taught a gene-editing course in Spring 2024 for two undergraduate and seven graduate students, covering molecular biology fundamentals, gene cloning, and CRISPR-Cas9 applications. Course learning outcomes included: Applying molecular biology concepts for guide RNA cloning. Defining CRISPR-Cas9 components and mechanisms. Comparing CRISPR-Cas9 with TALENs and ZFNs. Differentiating between germline and somatic editing in plants and animals. Evaluating CRISPR applications in research and biotechnology. Assessing ethical guidelines for CRISPR use. Communicating CRISPR-related findings effectively. Demonstrating gene-editing techniques in practical sessions.
Publications
- Type:
Peer Reviewed Journal Articles
Status:
Accepted
Year Published:
2023
Citation:
Roy, Sonali, et al. "The peptide GOLVEN10 alters root development and noduletaxis in Medicago truncatula." The Plant Journal 118.3 (2024): 607-625.
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Progress 05/01/22 to 04/30/23
Outputs
Target Audience:During this reporting period, our project targeted a variety of audiences, from graduate and undergraduate students, to legislatures, alumni, and peer scientists and researchers. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?This project provided training opportunities to two TSU graduate students and three TSU undergraduate students. Miss Divya Jain, a PhD candidate in Agricultural sciences at Tennessee State University is working on Medicago CAPE peptides and on Aim 1 of the proposed project. She completed her M.S degree in Agricultural Sciences last year and wrote the manuscript "Gene editing to improve legume-rhizobia symbiosis in a changing climate" as part of her degree. This was published in the invitation only journal Current Opinion in Plant Biology. For her dissertation research Miss Jain is looking at the molecular responses to application of synthetic peptides. She is looking at the changes in methylation patterns caused by application of synthetic peptides on nitrogen starved root tissue and roots infected with rhizobia. This is part of Objective 1 of this project. Together with a second graduate student, Ms. Jain has isolated DNA from peptide treated roots and this has been submitted to the company Novogene for the next sequencing steps. Mr. Manoj Kumar Reddy Allam is a graduate student studying towards an M.S in Agricultural Sciences. Manoj presented his research at the American Society of Plant Biologists Midwest conference in Ames, Iowa. During this degree he learnt molecular bioogy techniques, the role of peptides in symbiosis. He participated in extracting DNA from roots treated with various peptides to be sequenced for methylation (Objective 1). Undergraduate Students Undergraduate senior Miss Jazmine Norwood completed a project in PI Roy's lab during the spring semester. She graduated summa cum laude in May and was awarded a degree in B.S Agricultural Sciences. Miss Norwood is currently interning with the USDA over summer and plans to return to TSU to undertake a master's degree in Agricultural Sciences. Miss Norwood completed a project during which she tested the effect of the synthetic peptide PIP1 on its ability to induce senescence in leaves of the legume Medicago truncatula. Her project found that the CRISPR gene edited pip quadruple mutants were less sensitive or insensitive to the senescence induced by application of the PIP1 peptide. Norwood verbally presented her research to visiting delegates from Bayer and NIFA during the semester that she worked in Roy's lab. Undergraduate freshman Miss Kyla Hughes worked in PI Roy's lab for two semesters during Fall 2022 and Spring 2023. She has prepared a poster and a talk that will be delivered at the upcoming international society of Plant Microbe Interactions Congress 2023 in Providence, Rhode Island. Her project tests the effects of the synthetic peptide MtCAPE16 on root growth traits in Arabidopsis. This is a non-legume model plant widely used in plant science research to understand gene regulatory networks and in studying plant hormones. Miss Hughes next screened the T0 and T1 generation of Arabidopsis lines overexpressing the MtCAPE16 gene in Arabidopsis and identifying stably overexpressing lines that are now being screened for phenotypes. During this project Miss Hughes learnt about the importance of nitrogen, root architecture and the role of small signaling peptides in regulating these processes. She gained technical skills in DNA extraction from lead tissue, PCR, plasmid DNA extraction, and light microscopy. Miss Hughes plans to pursue a career in research and will continue to explore her interests over the next three years while at TSU. Kyla is an invited speaker to the IS-MPMI congress where together with graduate student Divya Jain, she will be presenting her research in a 10 minute talk in July 2023. Dr. Ali Taheri, Co-PI had one undergraduate student in his lab during the fall and spring semesters. Miss Janae Terrell learned the basic molecular tools and techniques and became familiar with PCR and bacterial transformation. At present, Miss Terrell is in a summer internship program at the University of Wisconsin working on similar projects. The lab continues to meet every week and students meet with PD Roy in one-on-one meetings every week. Roy has trained students in using the electronic lab notebook Benchling where the students record their progress. Roy taught three students how to make scientific posters to disseminate findings. She also trained students Divya Jain and Kyla Hughesin presenting talks at conferences. Roy has helped the two graduate students in preparing their CVs.Students and PD Roy work on SMART goals at the beginning of the year as well as a mentorship agreement that outlines goals and expectations of both, the PD and the student. This is reviewed at the end of the year or the project to gauge whether learning needs have been met. All student achievements are highlighted via the PD's social media pages as well as the newly created lab website legumegenetics.org. This project continues to provide a valuable platform for students to learn, grow, and contribute to the field of agricultural sciences. Our track record over the past year and our plans for the future reflect a steadfast commitment to training the next generation of agricultural scientists. How have the results been disseminated to communities of interest?Legislatives, alumni Our work has been recognized and highlighted in two substantial articles read by a significant number of alumni and legislators. The first, titled "Faculty Research Highlights - Sonali Roy", written by Celeste Brown and published in the 2022 Research Horizons magazine. Distributed by the Research and Sponsored Program Office, it serves as the annual magazine for legislators and alumni. This article outlines the NIFA grant and provides details of PD Roy's research program. The second article, "New asst. professor bears golden touch", authored by Joan Kite, was featured in the Summer 2022 edition of Ag Link, a college of agriculture magazine. Over 3000 copies of this magazine were distributed to alumni. Lastly, Roy was the olny faculty selected to present her research to the newly formed board of directors at the College of Agriculture. The board includes representatives from 10-15 institutions, industry partners such as Bayer and Corteva who will decide the overall directions and measure progress of the College. https://issuu.com/tennesseestateuniversity/docs/_9.26summeraglink2022/s/17075045 https://www.tnstate.edu/research-1/publications/annualreport.aspx 2022 Peer Scientists and Researchers: Manuscripts Jain D., Jones L.B., Roy, S.# (2022). Gene editing to improve legume-rhizobia symbiosis in a changing climate Current Opinion in Plant Biology Monaghan, M., Brady, S.M, Haswell, E.S., Roy S., Schwessinger, B., McFarlane, H.E (2022, Accepted). Running a research group in the next generation: combining sustainable and reproducible research with values-driven leadership Journal of Experimental Botany, https://doi.org/10.1093/jxb/erac407 Seminars Sonali Roy (Project Director) Invited Seminar - Pennsylvania State Univeristy:Peptides in Nodulation and Nitrogen Acquisition. (Happy Valley, PA, April 2023) Invited Plenary Speaker - ICRISAT 10th International Conference on Legume Genomics and Genetics - 2022 eSymposia:Genetic discoveries in legume nodulation and symbiotic nitrogen fixation. Virtual Seminar (Nov 2022) Invited Seminar - University of Missouri:Peptides in Nodulation and Nitrogen Acquisition. (Columbia, MO, September 2022) Invited Seminar - University of Vermont:Peptides in Nodulation and Nitrogen Acquisition. (Burlington, VT, September 2022) Invited Keynote Speaker - Donald Danforth Plant Science Center Scientific Retreat:Peptides in Nodulation and Nitrogen Acquisition. (St. Louis, MO, June 2022) Invited Seminar - North American Symbiotic Nitrogen Fixation Conference:Regulation of nitrogen acquisition by peptide hormones. (Madison, WI, June 2022) Divya Jain (Graduate Student) Concurrent Session: "CAPE Peptides are involved in Nitrogen Acquisition and Nodulation in Medicago truncatula,North American Symbiotic Nitrogen Fixation Conference, University of Wisconsin (2022). Oral talk,Tennessee Academy of Science, Annual Meeting, Tennessee State University (2022). Oral talk,Tennessee State University Annual Research Competition, First prize winner (2022). Manoj Kumar Reddy Allam (Graduate Student) Oral talk,Tennessee Academy of Science, Annual Meeting, Tennessee State University (2022). Poster, American Society of Plant Biologists Midwest conference, Ames, Iowa (2023) What do you plan to do during the next reporting period to accomplish the goals?During the forthcoming reporting period, we have a number of key goals we are aiming to achieve. The first of these is the completion of one manuscript, specifically related to the results achieved from objective 1. This will include drafting the findings, and also incorporating them into a comprehensive manuscript that details our methodology, analysis, and the implications of our results. This will not only contribute to the existing body of research in this field and provide a strong foundation for future studies but also provide training to the graduate student in drafting publishable scientific manuscripts. Next on our list is the design, cloning, and initiation of transformation for all four CRISPR-Cas9 constructs. This is a critical step towards manipulating and understanding the genome in our study. The graduate student starting in Fall 2023 will be trained in designing targeted gene sequences to clone into the CRISPR-Cas9 system, with the goal of altering the genes of interest. After cloning, we will initiate transformation procedures to incorporate these engineered sequences into cells, taking us one step closer to understanding their function and their impact on the overall objective. Furthermore, a major facet of our project lies in fostering the next generation of scientific researchers. To this end, we aim to welcome at least one more graduate student into our team. Oour team also plans to integrate two additional undergraduate students into our research framework. These students will gain invaluable exposure to the process of scientific research by completing hypothesis-driven work under my supervision.
Impacts
What was accomplished under these goals?
Objective 1 Progress: To quantify changes to the Medicago methylome we are conducting two large scale experiments. The first on roots starved of nitrogen in the presence and absence of synthetic peptides. We selected three peptides MtCAPE16, MtGLV10 and MtCEP1-D1. While there is significant information about the peptides GLV10 and CEP1D1 very little information is known about the role of MtCAPE16. Any information we thus find about the role of this peptide will be new information. Identifying peptides with biological activity: We started by characterizing the effects of CAPE16 on Medicago roots. Our research focuses on the biological activity of CAPE16, a Nitrogen-responsive CAP-derived peptide, in Medicago roots. Findings from synthetic peptide treatment based experiments indicated that exogenous application of synthetic MtCAPE16 altered root morphology based on nitrogen availability. In the A-17 ecotype, sufficient nitrogen saw a 47.46% reduction in lateral roots. Conversely, the sunn-4 seedlings saw increased lateral roots across all nitrogen conditions, with notable increases of 28.98% and 45.45% under low and sufficient nitrogen conditions respectively. Additionally, MtCAPE16 significantly increased root hair length across all nitrogen conditions in both A-17 and sunn-4 seedlings, confirming MtCAPE16's biological activity. For a deeper understanding of CAPE16's impact, we generated a gene edited A17 line using CRISPR-cas9. To understand the effect of CAPE16 on downstream processes, we generated a gene edited line in A17 wild type plants using CRISPR-cas9. Whole Genome Bisulphite Sequencing: To understand the effects of exogenously applied peptides we are conducting a WGBS of Nitrogen starved Medicago roots that have been treated with peptides. We germinated 1000 Medicago truncatula seedlings and grew them on water agarose plates with 1% agarose. After three days, seedlings were transferred in the morning to beakers with 100 ml of Broughton and Dilworth medium with and without Nitrogen. Care was taken to make sure roots were fully immersed in the solution and seedling leaves were floating on the liquid media. Approximately 80 -100 seedlings were transferred per beaker and the top sealed with saran film to prevent evaporation. Beakers were left in growth chambers at 24 C with a light regime of 16-hour light and 8 hour darkness. After 24 hours, seedlings were removed from their treatment solution and patted dry gently. Roots were excised using a sterile scalpel blade. Roots from twenty seedlings were pooled into one replicate. All roots were excised within fifteen minutes of the first sample. The samples include three peptides as mentioned before, one scrambled control peptide and one no-peptide control. Each treatment had four replicates with 20 seedlings each. Each of these sets was compared for with and without 6 mM nitrogen supplied as ammonium nitrate and potassium nitrate totaling 40 samples. DNA was extracted from these samples using the Qiagen DNA plant DNA extraction kit and treated with RNAse. The samples were run on a gel to determine the quality and that the DNA were not sheared. Thirty samples were then sent to Novogene for WGBS. A second set of samples comprising of nodules treated with peptides are currently under preparation. We have optimized in vivo conditions with rhizobia spot inoculated roots where 7 of 10 roots form nodules within seven days. Marking the site of inoculation leads to formation of nodules just below this site reproducibly allowing us to collect root tissue with emerging nodules not visible to the naked eye. We have had some trouble extracting sufficient amount of DNA for downstream processing. We are therefore now scaling up our initial sample size and the number of spot inoculated segments we will collect. Objective 2 Progress: I. Evaluation and reanalysis of single replicate RNAseq data treated with multiple peptides: We have focused our study on four key enzymes, two methyltransferases and two acetyltransferases, which have been selected for gene editing due to their significant expression changes post peptide application. We identified four enzymes that showed the highest change in gene expression in response to peptide application. These genes include: Methyltransferases: Medtr1g098000 (MtSDG15), Medtr1g098030, and Medtr1g098370; Medtr7g109560 (MtSDG63) Acetyltransferases: Medtr1g103110(MtHAG4), Medtr1g103250(MtHAG5); Medtr5g0064050(MtHAG24), Medtr5g006460 (MtHAG25), and Medtr5g006470 (MtHAG26) Our knockout strategy will include guide RNA (gRNA) design targeting homologous genes as well, to ensure complete and efficient knockouts without compensatory effects from related genes. The next phase of this project will be carried out by a graduate student joining our team in the fall. Objective 3 Progress:We are on track to train graduate students and undergraduate students. The project has made contributions to the graduate theses of one M.S student, Manoj Kumar Reddy Allam, and one Ph.D. student, Divya Jain. Training opportunities includes attendance at both national and international society meetings and regional meetings, exposing the students to the wider scientific community and broadening their perspectives. We anticipate the participation of additional graduate students from Fall 2023 onward. In addition, we have made considerable strides in fostering undergraduate academic involvement. We have successfully engaged three undergraduate students, all of whom are part of the prestigious Dean's Scholars program. To deliver on the goal of hypothesis driven short term research projects for undergraduate students as proposed here, we designed two peptide screening projects for the two undergraduate students trained in PD Roy's lab. Project 1 (Miss Jazmine Norwood): In collaboration with Dr. Kashif Mahmood at Oklahoma State University, we discovered through preliminary data that application of PIP peptides led to accelerated leaf senescence in Medicago. Our hypothesis for this project was that quadruple peptide mutants of Medicago (pip1pip4pip5pip7) would demonstrate decreased sensitivity to PIP1 peptide application. We conducted thorough experiments involving the application of pip1 peptide on these quadruple mutants, and we analyzed the effects on leaf senescence. The research process involved repeated experiments with over 18 individual plants, specifically testing leaf metamer2 and metamer3 from each plant. Contrary to our initial hypothesis, the application of the peptide did not reduce leaf senescence in the quadruple mutants. Instead, the results showed either no effect or an increase in the amount of chlorophyll detected in the leaves of the mutant plants, indicating no acceleration in leaf senescence. Each round of experimentation also included the wild-type control (R108), plants transformed with an empty vector as a control, and two different alleles of the pip quadruple mutants to ensure a comprehensive and unbiased evaluation of the effects. These findings provide the first evidence in literature supporting the involvement of PIP peptides in leaf senescence. Project 2 (Miss Kyla Hughes): Miss Hughes' research, inspired by Divya Jain's findings, investigated the MtCAPE16 peptide's role in lateral root development of Medicago truncatula. Given that MtCAPE16 doesn't have an orthologue in Arabidopsis, it was hypothesized that its application wouldn't impact Arabidopsis's lateral root development. Contrarily, applying MtCAPE16 increased Arabidopsis's lateral root count in two out of three experiments. Further, overexpression of MtCAPE16 was explored, with Hughes identifying three T0 and five T1 lines positive for the transgene. These lines are being phenotyped to understand MtCAPE16 overexpression's effect on lateral root development.
Publications
- Type:
Journal Articles
Status:
Accepted
Year Published:
2023
Citation:
Jain, Divya, Lauren Jones, and Sonali Roy. "Gene editing to improve legume-rhizobia symbiosis in a changing climate." Current Opinion in Plant Biology 71 (2023): 102324.
- Type:
Journal Articles
Status:
Accepted
Year Published:
2023
Citation:
Monaghan, Jacqueline, Siobhan M. Brady, Elizabeth S. Haswell, Sonali Roy, Benjamin Schwessinger, and Heather E. McFarlane. "Running a research group in the next generation: combining sustainable and reproducible research with values-driven leadership." Journal of Experimental Botany 74, no. 1 (2023): 1-6.
- Type:
Theses/Dissertations
Status:
Accepted
Year Published:
2022
Citation:
Divya Jain, M.S Agricultural Sciences (non-thesis), Tennessee State University
- Type:
Theses/Dissertations
Status:
Under Review
Year Published:
2023
Citation:
Manoj Kumar Reddy Allam, Investigating Nodule Glycine Rich Protein and GOLVEN10 Peptide Functions in Root Nodule Symbiosis and Legume Root Architecture Control
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