Progress 09/01/23 to 08/31/24
Outputs
Target Audience:In the third year of the project, our target audiences included a wide range of audiences, with the broadest groups being global/international researchers across all fields of biology and genomics and the narrowest being local public high school groups and the local community. Specifically, activities targeted the broadest groups globally through (1) development of our genomic data-mining software tool (SRAMiner) which is free and open source and can be used to rapidly search for any custom gene or genome target from petabyte-scale publicly available global DNA sequence databases, and (2) development of our gene annotation software tool (DarkMatter) which will be free and open source and will assign gene features for our specific project and any other project with genes lacking specific annotations. We also target broad researchers globally through (3) publishing a book chapter on methodologies for analysis of Wolbachia in plant-parasitic nematodes which will be useful for audiences across many fields of nematology, symbiosis, and microbiology. The project also targeted diverse scientists across diverse fields through (1) a seminar presentation to faculty, students, and researchers at The Institute of Environmental & Human Health at Texas Tech University (TTU TIEHH), (2) a presentation at the American Society of Microbiology Conference, (3) a presentation at the joint conference of American Society of Agronomy (ASA), Crop Science Society of America (CSSA) & Soil Science Society of America (SSSA), (4) a presentation to the Society of Nematologists Conference, and (5) a presentation at the Society for Advancement of Chicanos/Hispanics and Native Americans in Science (SACNAS) National Diversity in STEM (NDiSTEM). I targeted local graduate students through (1) formal classroom courses including two courses (Microbiomes and Bioinformatics) integrating original research related to this project, (2) formal conventional courses (Advanced Microbiomes) that involved research presentations related to this project, (3) research experiences in my lab for graduate students both as major advisor and as a directed studies advisor, and (4) having my graduate mentees present their research in three presentations at a local research conference. I targeted local undergraduate students through (1) formal classroom courses including two courses (Microbiomes CURE and Bioinformatics CURE) integrating original research related to this project, (2) formal conventional courses (undergraduate Microbiomes) that involved research presentations related to this project, (3) research experiences in my lab for Biology Major, Computer Science Major, and Microbiology/Chemistry Major students integrating this project into the research, (4) peer-mentoring-based summer intern research programs such as the project-funded Bioinformatics Challenge program and the Genomics Scholars program, and (5) having my undergraduate mentees present their research in three presentations at the local Texas Tech Undergraduate Research Conference. I targeted local high school students through the project-supported Genomics Scholars program which involved bringing local students to my lab to perform research in peer-based mentoring teams, carrying out research related to this project. I targeted racial and ethnic minorities and those who are socially, economically, or educationally disadvantaged though supporting the intensive >2-year long research mentees from the NIH-affiliated "Bridges to the Baccalaureate" program (for students transitioning from 2-year colleges) and the NSF-affiliated Louis B. Stokes program (for underrepresented minorities). I also reached out to underrepresented high school students in recruiting local students for the Summer Genomics Scholars program in my lab. We also had one of our trainees present at the SACNAS National Diversity in STEM (NDiSTEM) Conference. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?Training Activities: In this project year, the project year provided training opportunities for 4 PhD students (directly mentored by me) plus 10 additional graduate students (through my research-based courses) (= 14 graduate students in total). The project also provided training for 8 undergraduate students (directly mentored by me and my graduate students), and 10 additional undergraduate students (through my research-based courses (= 18 undergraduate students in total). We also trained 5 high school summer research students. Directly Mentored PhD Students: My graduate student Amandeep Kaur (PhD Candidate) was trained in scientific writing towards her first publication. She also gained advanced training in bioinformatics analysis this year, and continues to acquire advanced mentorship in genomic library preparation and new methods in nematode culturing. She has also been learning how to lead teams of high school Genomics Scholars supported through this project. My grad student Taranjot Kaur (previously an MS student with me who has moved on to do a PhD in my lab) has been mentored by me and other advanced students in techniques in genomics, bioinformatics, and manuscript writing, with her first paper submitted. She has also gained training in mentorship of undergraduates. My grad student Era Sharma (PhD student) has been trained in advanced computer programming - particularly for handling Big Data analysis, with highly parallelized processing. She has gained additional skills in writing her first paper for publication. She continues training in mentoring undergraduate computer science majors as part of the Bioinformatics Challenge program supported by this project. My student Shiva Aghdam (PhD Candidate) was trained in confocal microscopy and HiC as well as aspects of lab management and project management. Additional Mentored Grad Students: The project activities supported integrated research and education for 10 graduate students through my research-based graduate courses, titled "Microbiomes Research Methods" (1 student), "Bioinformatics of Agricultural Microbiomes" (4 students), and "Advanced Microbiomes" (5 students). Each of these courses involved synchronously taught undergraduate active learning and research projects, with skills-training in project design, statistics, genomics library preparation, DNA analysis methods, oral and written communication, and research best-practices training. Directly Mentored Undergraduate Students: This year I mentored 8 undergraduate students in research related directly to this project, with the help of my graduate students. These included biology/chemistry majors Rylee Dallison and Emmalee Rose who were trained in nematode isolation, PCR, microscopy methods, and genomics. Trainings were also performed for Kyle Walker, recruited from my CURE course to develop skills in Citizen Science program and materials development. We continued to train Angela Marentes and Faith Ruckey in plant-parasitic nematode culturing for creation of iso-female lines. Faith also gained training along with Shanthan Sudhini, Rohit Chabukswa, and Gargi Pathak in advanced computer programming and mathematical modeling through my Bioinformatics Challenge summer program supported by this project. Additional Mentored Undergraduate Students: My lab also trained an additional 10 undergraduate students in research associated with this project through my research-based CURE courses (titled "Microbiomes CURE", "Bioinformatics CURE", and "Microbiomes - HEPlus"). Students developed skills in molecular biology and fundamental skills in analysis and scientific experiment design. High School Mentees: This project year supported mentoring for 5 high school summer research students from local Lubbock high schools with diverse student populations. We worked directly in the laboratory for most of the summer with Carolina Acosta (High School Genomics Scholar) working on culturing and data analysis, Jamie Kim (High School Genomics Scholar) working on Baermann funnel extraction and PCR primer design, Shirley Chen (High School Genomics Scholar) working on nematode DNA isolation and PCR, and Mary Kim (High School Genomics Scholar) working on protocols for Wolbachia transection into other nematodes. We also mentored William Xie (Clark Scholar) in nematode extraction and computational approaches to comparative genomics of endosymbionts. Professional Development: PhD student Amandeep Kaur gained training and experience publishing a first-author book chapter. She also gained skills in presenting at one national conference (Society of Nematologists) and one local conference. PhD student Era Sharma gained experience in writing her first manuscript for a peer-reviewed journal for her software (fully uploaded to GitHub), and presenting her work at a local conference. PhD student Taranjot Kaur gained skills in presenting her work at two major conferences (ASM Microbe and ASA, CSSA, SSSA Conference), as well as a local conferences. She also gained training in her first-author manuscript writing and submission. Undergraduates Rylee Dallison, Faith Ruckey, and Angela Marentes gained valuable training in presenting their work at a local conference (Texas Tech University Undergraduate Research Conference). Faith also gained skills in scientific communication by presenting her project work at SACNAS. How have the results been disseminated to communities of interest?Results of this work were presented through an invited talk by PI Brown to diverse members of the Institute of Environmental & Human Health at Texas Tech University (TTU TIEHH), in a talk titled "Mining DNA and RNA at the plant-animal-microbe interfaces to solve the puzzles of symbiosis". Results were also disseminated through our published book chapter (Kaur and Brown, 2024 "Detection and analysis of Wolbachia in plant-parasitic nematodes and insights into Wolbachia evolution" in the book "Wolbachia - Methods and Protocols", ed. A. Fallon. Springer Nature. We anticipate will be widely useful to diverse researchers across fields of insect and nematode science and in the field of symbiosis/parasitism. We anticipate a wide global audience of scientists from diverse fields in agriculture and other life sciences through our two project-related software programs under development. The program "SRAMiner" that is completed and available on GitHub free and open source has been appreciated at conference presentations (e.g., a talk by Sharma E (presenter) and Brown AMV. "Development of an automated NCBI SRA mining tool for target discovery" presented at the Texas Tech Annual Biology Student Symposium (TTABSS), Feb. 9-10, Lubbock, TX.). Our second software tool, titled "DarkMatter" is at an earlier stage of development, but has been of interest to various faculty and students when discussed or presented through classroom or Bioinformatics Challenge program meetings. An important outreach effort made in this project year is the interactions with several campus programs for students from underrepresented groups in STEM. These include meetings and program ads/recruitment interviews with students from the Black Students' Society, the Hispanic Students Society, Women in Computer Science, and Texas Tech's "First-Gen" Mentoring program for first generation college students. Our undergraduate lab member Angela Marentes is part of the "Bridges Across Texas - Louis Stokes Alliance for Minority Participation" and our lab member Faith Ruckey is a member of the "NIH-supported Plains Bridges to the Baccalaureate Program". Faith presented work from this project at the Society for Advancement of Chicanos/Hispanics and Native Americans in Science (SACNAS) National Diversity in STEM (NDiSTEM) Conference on Oct. 26-28 2023 in Portland, OR. Further results have been disseminated in talks and posters by students supported by this project, including: -a talk at the Texas Tech Annual Biology Student Symposium (TTABSS) in 2024 by Taranjot Kaur titled "Transcriptomic assessment of the effects of the sorghum root exudate, sorgoleone, on root-lesion nematodes" Feb. 9-10 in Lubbock, TX. -a talk at the Texas Tech Annual Biology Student Symposium (TTABSS) in 2024 by Amandeep Kaur titled "Wide geographic survey with new PCR screen reveals plant-parasitic nematodes host diverse hidden endosymbionts" Feb. 9-10, Lubbock, TX. -a poster at the American Society for Microbiology (ASM Microbe) meeting in 2024 by Taranjot Kaur titled "Discovery of novel Wolbachia strain in Heterodera expands nematode host distribution" Jun. 13-17 in Atlanta, GA. -a poster at the American Society of Nematologists meeting in 2024 by Amandeep Kaur titled "High prevalence of Wolbachia and Cardinium revealed in different geographical populations of plant-parasitic nematodes" Aug. 4-9 in Park City, UT. -two posters at the Texas Tech Undergraduate Research Conference in 2024: one presented by Ryleee Dallison titled "Analysis of endosymbiont prevalence reveals unexpectedly widespread abundance in nematodes" Apr. 9-10, Lubbock, TX; and one presented by Angela Marentes titled "Exploring bacterial endosymbionts Wolbachia and Cardinium in plant-parasitic nematodes" Apr. 9-10 in Lubbock, TX. -a poster at the joint meeting of the American Society of Agronomy (ASA), Crop Science Society of America (CSSA) & Soil Science Society of America (SSSA) in 2023 by Taranjot Kaur titled "Transcriptome assessment of the effects of the sorghum root exudate, sorgoleone, on root-lesion nematodes" Oct. 29-Nov 1 in St. Louis, MS. Finally, some work and outcomes of this project were presented in research-centered classroom lectures to majors and non-majors at Texas Tech University. What do you plan to do during the next reporting period to accomplish the goals?During the next reporting period we will continue to advance the bioinformatics expertise of our team to achieve the remaining goals of the project. Currently, we have 3 PhD students dedicated to the project and one expected postdoc, plus a steady team of computer science and biology undergraduates with major roles in the project. We expect to make major breakthroughs and complete the remaining goals of Obj. 1, and more of the complex aspects of Obj. 2 and Obj. 3, as well as continue to bring 4 manuscripts in preparation to completion for submission to peer-reviewed journals. Specifically, for Obj. 1, we expect to assemble complete genomes of 12 Wolbachia strains and 22 Cardinium strains from plant-parasitic nematodes by resequencing the samples at a higher coverage. We will combine these data with newly discovered strains from data mining (~40 further complete endosymbiont genomes). We expect to comparatively analyze complete endosymbiont genomes using pangenomic analyses, functional analyses (pathway analysis and gene ontology enrichment analysis) and detect function through de Bruijn genome-wide association study (DBGWAS) analyses, combined for signatures of selection (dN/dS) that will inform their roles in PPNs and formulating future strategies related to symbiont-based biocontrol of PPNs. A key goal in the year ahead for Obj. 1.2 is to finalize and submit the SRAMiner research paper for publication and make public the GitHub repository containing the tool, alongside detailed documentation and tutorials. For the DarkMatter software project, we will continue with the pipeline enhancements that transition the data processing to Apache Spark for real-time data handling and scalability. We will work on database migration from JSON data to a PostgreSQL database for improved data accessibility and analysis efficiency. Finally, we will implement Machine Learning Integration, initiating a phase of design of machine learning models for dynamic pattern recognition and prediction within genomic datasets. For Obj. 2, we plan to perform transcriptomic analysis of Pratylenchus penetrans populations (obtained from isofemale lines grown in the lab) with varying endosymbiont statuses to investigate their responses to temperature stress. This study will involve RNA sequencing to understand the differential gene expression patterns among populations harboring endosymbionts Wolbachia and/or Cardinium at a series of temperatures and under a variety of plant host conditions/exposures. Through this experiment, we aim to identify key genes and pathways involved in the tolerance to temperature stress and to uncover the potential role of endosymbionts in increasing host's stress responses. In addition, we hope to submit a manuscript on improved nematode culturing procedures. To support the nematode culturing and overcome major challenges with the very long generation times of Pratylenchus penetrans, we will continue with our microinjection (transfection) experiments to inoculate Wolbachia and Cardinium into other nematodes, including C. elegans. If successful transfections can be achieved, the outcomes should help in future steps towards exploring these symbionts for biocontrol. We plan to test the above functional effects in alternate hosts using RNA-seq, and stressors of temperature and plant root exudates to investigate host-symbiont interactions. For Obj. 3, Obj. 3.1, we will compare control and treatment greenhouse plant communities at different temperatures (passive heating shield) with nematode inocula (with and without symbionts) to controls (no nematodes), testing two-way effects of the interactions. Specifically, we will test the tripartite interactions between endosymbionts, PPNs, and plants using metatranscriptomics, fitness assays, and plant physiological/pathogenicity metrics. For Obj. 3.2, we will refine our foundational mathematical model and continue to convert the model into python code for running tests on both empirical experimental data and simulated data. We will run our Bioinformatics Challenge and Genomics Scholars programs again, with greater efforts to structure and organize the mentorship and evaluate its effectiveness formally, with assistance from Texas Tech's Center for Teaching and Learning. In our team take our Rhizosphere Citizen Science materials and run this program at sites across the U.S. We will integrate this program with aspects of our planned Rhizosphere Biomodelling Network outreach program, which is currently under development. I have continued to search for a suitable postdoc and will increase my efforts to advertise and recruit a strong postdoc to assist with leading advanced bioinformatic and modeling tasks.
Impacts
What was accomplished under these goals?
Key Impacts: Because plant-parasitic nematodes (PPNs) cause significant losses to crop yields and are extremely difficult to control, our efforts have focused on assessing ways that their bacterial endosymbionts function to impact PPN hosts in ways that may lead to sustainable biocontrol. Our successes include discovery and analysis of new endosymbiont and nematode genomes and transcriptomes, diversity profiling, prevalence assessment, cultures grown and tested in planta at different temperatures, and the development of software tools and modeling, as well as outreach and education detailed below: Obj. 1.1 Analyze population genomic data from field-sampled PPN populations for signatures of selection on endosymbiont genes. (1) Activities: We processed 65 samples collected from wild and farm locations. We performed dilution-PCR to select the best 56 samples for genome sequencing. We tested new nematode-specific PCR primers to amplify a ~583 bp region of 18S rRNA gene. For DNA samples in which universal bacteria primers did not produce a band, we used a test for inhibitors and then used a standard AMPure bead clean up protocol to purify inhibitors from the sample. (2) Data: Prevalence assays were performed for 11 samples (24 to 105 individuals each) using single-worm lysis. Sample P488 Ppen had 41.9% Wolbachia, 82.9% Cardinium, and 37.1% co-infected. P488 non-Ppen showed 67.7% Cardinium. P462 had no Wolbachia, 32.3% Cardinium. P464 had 12.5% Wolbachia, 26% Cardinium, and 7.3% co-infection, while P467 had 1.0% Wolbachia, 18.8% Cardinium, and 1 % co-infections. Male Ppen had exceptionally high infection rates. Genomes are currently under analysis. (4) Key Outcomes: Our genomic and 16S/18S analysis provided valuable insights and our PCR assay was included in a published book chapter authored by my PhD student. We successfully assembled two nearly complete Wolbachia genomes, providing valuable insights into gene repertoire composition and trained a large team of undergraduates and high school students in molecular biology, genomics, and bioinformatics. Obj. 1.2 Analyze drivers of evolution across global populations of PPNs and rhizosphere samples through bioinformatic database mining. (1) Activities: We performed small-scale and large-scale database-mining analyses for endosymbionts of PPNs from public datasets. In small-scale analyses, we screened 109 genomic sequence runs (SRRs) from 11 economically important PPN pests. We improved the python code to greatly improve the functionality of our software tool ("SRAMiner") for NCBI sequence read archive (SRA) screening, integrating MPI. To characterize PPN/endosymbiont function, we developed another software tool ("DarkMatter") with 3 components: 1: Alignment and Clustering of Sequences into Homologous Blocks; 2: Sequence Content Analysis in Homologous Groups; 3: Metadata download and mining. (2) Data: Small-scale data mining revealed 60 SRRs with Cardinium. Large-scale data mining using SRAMiner on 6,500 SRAs from diverse sources, including 14 species of PPN, revealed 352 Wolbachia-positive runs. DarkMatter software was tested on a toy set of 33 genomes, clustering homologs and using KaKs, GC, tetranucleotide, and PostgreSQL database integration facilitated metadata-querying capabilities. (4) Key Outcomes: Analysis showed symbionts clustered with known PPN-type strains, forming deep branches suggesting an undiscovered diversity. The first draft of the SRAMiner v4 manuscript has been completed, and the tool's GitHub page is fully prepared for release. DarkMatter software appears to be promising and on-track to become of broad value for novel gene annotation for the project. Obj. 2.1 Analyze Wolbachia and Cardinium endosymbiont effects on PPNs using root-cultures and measure fitness and microbe-host gene expression in these lines. (1) Activities: We improved carrot disc cultures using Gamborg's B5 in pluronic media. After 3 months from inoculation, Ppen were extracted. We began experimental transinfection of Wolbachia from plant-parasitic nematodes into C. elegans for further analysis of endosymbiont effects. (2) Data: Ppen adults and juveniles were recovered from parent plates and inoculated on new plates incubated at 25°C to produce 280 isofemale and 4 juvenile plates. We successfully recovered from 3 to over 300 individuals from each isofemale line after 3 months. Yield was higher at 25°C than 26°C. Recovered isofemale lines were individually inoculated into soil pots containing cucumber plants to further increase population numbers for subsequent studies. (4) Key outcomes: We were successful in further optimizing Ppen culturing and temperature trials leading up to controlled fitness and transcriptome experiments to evaluate endosymbiont impacts toward developing biocontrol approaches. Obj. 2.2 Assess endosymbiont effects on PPN parasitism on greenhouse plants and transparent soils. (1) Activities: Nematode communities from diverse Wolbachia/Cardinium-positive and -negative were inoculated into greenhouse plants. We tested the effects of plant exudates on PPN gene expression (RNA-seq). (2) Data: RNA-seq results showed that the root exudate sorgoleone impacts gene expression in Pratylenchus scribneri with upregulation of 72 genes and downregulation of 54 genes, affecting processes like chromatin assembly, nucleosome assembly/organization, DNA packaging, chromatin remodelling, DNA conformation, and stress responses. We predict enhanced apoptosis in RLN populations may cause decrease in P. scribneri density in sorghum fields as a response to its exudate. (4) Key outcomes: Overall, it appears that sorgoleone caused stress in P. scribneri. We are expanding these experiments to other Pratylenchus species with and without endosymbionts. Obj. 3.1 Test endosymbiont effects on PPN parasitism in plant communities transplanted to the greenhouse under temperature stress. (1) Activities: We expanded the protocols and design for these experiments, based on the discoveries listed in the previous sections. (4) Key outcomes: These experiments are underway. Obj. 3.2 Develop and evaluate a computational model that probes endosymbiont-PPN-plant interactions under land-use and climate changes. (1) Activities: Summer Bioinformatics Challenge students Rohit Chabukswa and Faith Ruckey worked on mathematical modeling and framing the pseudocode for this objective. I worked to interview postdocs for this part of the project for the year ahead. (4) Key outcomes: The initial model framework for assessing endosymbiont-PPN-plant interactions is built. Edu Obj. 1: Develop a "Bioinformatics Challenge" Undergraduate Fellowship. (1) Activities: We successfully ran this program following the model of the previous year, recruiting diverse students through campus organizations. (2) Key outcomes: Participants produced code and models, supporting the computational and modeling outcomes described above. Edu Obj. 2: Create a "Genomics Scholarship" peer-to-peer mentoring for high schoolers from underrepresented minorities. (1) Activities: We ran a successful program, recruiting and training diverse high school students, as described in the previous year. (4) Key outcomes: Participants were trained in advanced genomics and bioinformatics approaches and produced outcomes described in the research sections above. Edu Obj. 3: Develop and pilot Rhizosphere Citizen Science and Rhizosphere Biomodelling Network programs. (1) Activities: A student from my CURE class contributed detailed materials and procedures for our Rhizosphere Citizen Science program. I again advertised for a postdoc whose role will include helping to lead the Rhizosphere Biomodelling Network program. (4) Key outcomes: The Rhizosphere Citizen Science program is under development for full-scale launch in the year ahead.
Publications
- Type:
Book Chapters
Status:
Published
Year Published:
2024
Citation:
2024 Kaur A, Brown AMV. Detection and analysis of Wolbachia in plant-parasitic nematodes and insights into Wolbachia evolution. In Wolbachia - Methods and Protocols , ed. A. Fallon. Springer Nature. DOI: 10.1007/978-1-0716-3553-7 ISBN 978-1-0716-3552-0
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2024
Citation:
2024 Kaur T (presenter), Brown AMV. Discovery of novel Wolbachia strain in Heterodera expands nematode host distribution. ASM Microbe, Jun. 13-17, Atlanta, GA.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2024
Citation:
2024 Kaur A (presenter), Brown AMV. High prevalence of Wolbachia and Cardinium revealed in different geographical populations of plant-parasitic nematodes. Society of Nematologists Meeting, Aug. 4-9, Park City, UT.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2023
Citation:
2023 Ruckey F (presenter), Kaur A, Brown AMV. PCR screening of plant-parasitic nematode communities for Wolbachia. SACNAS National Diversity in STEM (NDiSTEM) Conference, Oct. 26-28, Portland, OR.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2023
Citation:
2023 Kaur T (presenter), Brown AMV. Transcriptome assessment of the effects of the sorghum root exudate, sorgoleone, on root-lesion nematodes. ASA, CSSA & SSSA International Annual Meeting, Oct. 29-Nov 1, St. Louis, MS.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2024
Citation:
2024 Marentes A (presenter), Kaur A, Brown AMV. Exploring bacterial endosymbionts Wolbachia and Cardinium in plant-parasitic nematodes. 16th Annual TTU Undergraduate Research Conference, Apr. 9-10, Lubbock, TX.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2024
Citation:
2024 Dallison R (presenter), Kaur A, Ruckey F, Brown AMV. Analysis of endosymbiont prevalence reveals unexpectedly widespread abundance in nematodes. 16th Annual TTU Undergraduate Research Conference, Apr. 9-10, Lubbock, TX.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2024
Citation:
2024 Kaur T (presenter), Brown AMV. Transcriptomic assessment of the effects of the sorghum root exudate, sorgoleone, on root-lesion nematodes. Texas Tech Annual Biology Student Symposium (TTABSS), Feb. 9-10, Lubbock, TX.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2024
Citation:
2024 Kaur A (presenter), Brown AMV. Wide geographic survey with new PCR screen reveals plant-parasitic nematodes host diverse hidden endosymbionts. Texas Tech Annual Biology Student Symposium (TTABSS), Feb. 9-10, Lubbock, TX.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2024
Citation:
2024 Sharma E (presenter), Brown AMV. Development of an automated NCBI SRA mining tool for target discovery. Texas Tech Annual Biology Student Symposium (TTABSS), Feb. 9-10, Lubbock, TX.
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Progress 09/01/22 to 08/31/23
Outputs
Target Audience:Target Audiences: The activities of the second year of the project reached numerous attendees at talks and conferences and in the local, national, and global arenas of science and agriculture. Presentations and direct training of students in the lab and classrooms at Texas Tech University (TTU) reached diverse target populations representing the demographic profile typical at TTU, which is a Hispanic Serving Institution with a large percentage of first-generation college students and students with rural backgrounds. Students most closely trained and engaged in the research included 2 PhD students and 1 MS student (100% female), and 13 undergraduates (8 female, 4 Black, 3 Hispanic), and 8 high school students. Efforts: Efforts made to reach target audiences include presentations and publications on the research methods (e.g., we submitted a book chapter on our methodologies) and outcomes of this work both by conference and seminar talks and in formal classroom situations (in PI Brown's class, Microbiomes), and in PI Brown's course based undergraduate research experience (CURE) course (Frontiers in Host-Microbe Genomics). Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?Training Activities: This project year provided training opportunities for 3 graduate students, 13 undergraduate students, and 8 high school student who focused on aspects of this project. The first graduate student (Amandeep Kaur, PhD student) has gained training in designing and tested a modified screening PCR assays for detection of endosymbionts from bulk nematode communities. She has developed new skills in working on single nematode PCR assays. She continues her training in bioinformatics. She has also gained skills in genomic library preparation, and nematology methods including working to generate live cultures. She has also been trained this year in publishing (a book chapter) and presenting her work effectively at conferences. Amandeep also gained skills this year in mentoring a larger team of undergraduates and high school students as part of the Genomics Scholars summer program. The second graduate student focused on this project (Taranjot Kaur, MS student - graduated in Summer 2023) has become trained in nematology, genomics, bioinformatics, and development of specific laboratory experiments on P. penetrans using the NemaLife physiological assay platform (in the engineering lab of Dr. Siva Vanapalli at TTU) and HPLC to isolate plant exudates to investigate the effects of root exudates on the nematodes. Taranjot has also gained skills in presenting her work effectively at conferences and drafting her work for publication. The third graduate student focused on this project (Era Sharma, PhD student) began in January 2022, and has focused primarily on bioinformatics goals of the project. She has gained advanced training in bioinformatic software development, primarily using python coding, with work in git, bash, and specific approaches to optimize multi-threading to optimize run time. She has assisted in lab work and has been gaining skills for further objectives of her PhD. Era has been trained in presenting her work effectively at conferences and has gained skills this year in mentoring a large team of undergraduate computer science majors as part of the Bioinformatics Challenge program supported by this project. Among the 13 undergraduate trainees who performed research focused on this project were Grayson Ressler who gained experience in Hi-C and microscopy and trained high school students, and Rene Zambrano who gained skills in PCR primer design and DNA extraction and PCR. Undergraduates Niyati Babaria and Nayalie Ozuna gained skills in nematode extraction and isolation from bulk communities. Students Rylee Dallison and Faith Ruckey have invested significant time in the research project and have gained skills in all molecular methods and nematode handling methods. They also gained experience in presenting their work at lab meetings and/or conferences. Student Kabuyi Ndaya gained skills in outreach, working to introduce the laboratory and project to students at local high schools. The remaining 6 undergraduates trained by the project were part of the "Bioinformatics Challenge" Program supported by this project. These students were divided into 3 teams of 2: students Nathan Pierce and Isaac Doggett were trained in bioinformatic coding skills as part of developing the novel software tool SRAminer v2 for NCBI sequence read archive data mining. Students Ayobami Ajala and Allison Saenz gained skills in programming as they worked on the first units of the novel software tool DarkMatter v1 which seeks to characterize unannotated genes based on metadata and signatures of selection. Sydney Rash and Zelepe Coulibaly gained python and HPCC skills in working on the latter units of the DarkMatter v1 tool. The 8 high school students who worked as part of the Genomics Scholars Program supported by this project had uniquely valuable training in our lab and gained a wide array of skills in laboratory methods and/or bioinformatics methods. Senior high school students Olivia Smith, Trista Arnold, and Mary Kim gained skills in handling nematodes communities, extracting DNA, and performing PCRs. Students Bobby Stephens and Savanna He gained skills in preparing genomic libraries for Hi-C and Lysis-Hi-C. Students Jason Ye and Zafeer Arefeen gained skills in microscopy (light microscopy and FISH) and working with data record-keeping and basic molecular biology instruments. Student Aaron Li gained skills in working on the HPCC and using metabolic modeling software and databases as part of the PathwayTools suite. He also gained skills in presenting his work and writing a report. Professional Development: PhD student Amandeep Kaur gained professional development experience through working to submit her first-author book chapter on methodologies that she developed. She also gained professional skills through presenting her work in talks and posters at conferences, including (1) at the 61st Annual SON Conference, Sep 26-30, 2022, in Anchorage, AK, (2) the Plant and Animal Genomes Conference XXIX, Jan 13-18, 2023, in San Diego, CA, (3) the Society of Nematologists Meeting, Jul 9-14, 2023, in Columbus, OH, and (4) the Texas Tech Annual Biology Student Symposium (TTABSS), Mar 31-Apr 1, 2023, in Lubbock, TX. MS student Taranjot Kaur gained professional development experience through working in an interdisciplinary setting with chemists/engineering labs, gaining new skills outside those used in our lab. She also gained professional experiences through presenting her work in talks and posters at conferences, including (1) at the Plant and Animal Genomes Conference XXIX, Jan 13-18, 2023, in San Diego, CA, (2) at the Texas Tech Graduate School Poster Competition, Mar 30, 2023, in Lubbock, TX, and (3) at the Texas Tech Annual Biology Student Symposium (TTABSS), Mar 31-Apr 1, 2023, in Lubbock, TX. PhD student Era Sharma gained professional development experience through presenting her work as a poster at the American Society of Microbiologists (ASM) Microbe, Jun 15-19, 2023, in Houston, TX. How have the results been disseminated to communities of interest?Some results of this work were presented by PI Brown to her department including to faculty, graduate, and undergraduates with a wide range of interests at Texas Tech University in Lubbock, TX, on Sep. 21, 2022, with a seminar titled "Paradoxically together: theory, discoveries, and new methods in analysis of emerging and long-term mutualisms". Additional results were presented by PI Brown to an audience with various areas of interest in agriculture in an invited seminar to the Department of Botany and Plant Pathology at Purdue University in West Lafayette, IN, on Oct. 19, 2022, with a talk titled "Comparative genomics of endosymbionts Wolbachia, Cardinium, and Xiphinematobacter with potential to control plant-parasitic nematodes". PI Brown presented other results from the project this year on Feb 15, 2023, to the program directors and awardees of the National Science Foundation - National Institute of Food and Agriculture Plant Biotic Interactions Program Awardee Meeting (Virtual). PI Brown also presented results at a major international scientific meeting on Jun. 11-16 in Crete, Greece at the 11th Wolbachia Conference, in a talk entitled "Widescale sampling and analysis of plant-parasitic nematode Wolbachia uncovers new strains and unsolved puzzles." Further results have been disseminated in talks and posters by the graduate students supported by this project, including: -an oral presentation in 2023 by Taranjot Kaur titled "Discovery of a novel Wolbachia strain associated with sugarbeet nematode suggestive of ancestral symbiosis" at the Texas Tech Annual Biology Student Symposium (TTABSS) on Mar. 31-Apr. 1, Lubbock, TX. -an oral presentation in 2023 by Amandeep Kaur titled "Bioinformatics analyses reveals diverse genomes of Cardinium endosymbiont in soybean cyst nematode" at the Texas Tech Annual Biology Student Symposium (TTABSS) on Mar. 31-Apr. 1, Lubbock, TX. -a poster presentation in 2023 by Era Sharma titled "Analyzing hidden symbionts in sequence archives using a novel bioinformatic database mining software tool" at the American Society of Microbiologists (ASM) Microbe, Jun 15-19, Houston, TX. -a poster presentation in 2023 by Amandeep Kaur titled "Unearthing the hidden world: exploring the endosymbionts in nematode communities through soil sampling" at the Society of Nematologists Meeting, Jul 9-14, Columbus, OH. -a poster presentation in 2023 by Taranjot Kaur titled "Discovery of a novel Wolbachia strain associated with sugarbeet nematode suggestive of ancestral symbiosis" at the Texas Tech Graduate School Poster Competition, Mar. 30, Lubbock, TX. -a poster presentation in 2023 by Amandeep Kaur titled "Comparative genomic analyses and enrichment test suggest evolutionary specialization of Cardinium symbiont" at the Plant and Animal Genomes Conference XXIX, Jan 13-18, San Diego, CA. -a poster presentation in 2023 by Taranjot Kaur titled "Discovery of putative Wolbachia HGT in the sugarbeet nematode suggestive of ancestral symbiosis" at the Plant and Animal Genomes Conference XXIX, Jan 13-18, San Diego, CA. -a poster presentation in 2022 by Amandeep Kaur titled "Bioinformatic analysis of Cardinium symbionts in soybean cyst nematodes" at the 61st Annual SON Conference, Sep 26-30, Anchorage, AK. Finally, some results were disseminated through presentations of work in progress by the PI and graduate students in the courses "Microbiomes" and "Frontiers in Host-Microbe Genomics" comprising approximately 68 undergraduates and 12 graduate students with a broad range of degree of study majors or fields of interest at Texas Tech University. What do you plan to do during the next reporting period to accomplish the goals?During the next reporting period, with the increasing skills of our team, including three graduate students (two PhD students and one just graduated MS student who decided to continue with our lab for her PhD), we expect to make major inroads on completing core goals of objectives 1.1 and 1.2, including fully sequencing, assembling, and comparatively analyzing endosymbiont genomes for signatures of selection that will inform future strategies related to symbiont-based biocontrol of PPNs. Given the inroads we have made in culturing and collectings distinct populations of P. penetrans, we expect to continue to complete steps towards objective 2.1 in the year ahead, obtaining initial data by the end of spring/summer. We will continue to build these stable cultures so that we can then perform key assays in transparent soils as per objective 2.2 in the following year. As described above, we found our PCR assay was sensitive enough to detect unexpectedly low titer symbionts in these samples, leading us to expand aspects of our PCR survey to reduce unnecessary costs of sequencing. In the year ahead, we hope to publish a manuscript describing these assays. The assays also facilitate further study of prevalence within populations via single-nematode PCRs, however, for applicability and practicality, we hope to move from serial dilution PCR to qPCR that accomplishes the same objective on trace targets. So, in the year ahead, we plan to develop and test this qPCR assay paralleling our end point PCR assay. We expect to limit further field sampling to locations from which we obtained positive samples. For those locations, we will re-sample host soils and roots/plants to carry out trials for Obj 3.1 and 3.2. In parallel with aposymbiotic trials above, we will set up and assess our first temperature tests in native transplanted grasses and turfgrass collected from a university-associated research plot near campus which we found to have P. penetrans. We expect to be able to run our Bioinformatics Challenge and Genomics Scholars programs again in the year ahead, with some minor revisions in our approach to ensure the best outcomes possible. With these programs running effectively, we will take steps forward in the building our Rhizosphere Citizen Science and Rhizosphere Biomodelling Network outreach programs. With new postdoc applicants, I hope to select and hire a suitably trained postdoc to assit with some of the more advanced bioinformatic and modeling goals of this project. Alternatively, since we have a PhD student who is very strong in bioinformatics, I will consider reaching out more broadly for postdoctoral applicants with mathematical modeling skills.
Impacts
What was accomplished under these goals?
IMPACT: This year, our team including PI Brown with 3 graduate students, 13 undergraduate students, and 8 high school students processed hundreds of samples from the previous sampling year and developed a new highly sensitive PCR assay and screened most of these populations to find a rich diversity of Wolbachia and Cardinium endosymbionts. We successfully established nematode cultures and media for temperature experiments. We tested plant exudate effects on nematode and symbiont transcriptomes. We developed bioinformatic tools for big data mining for PPN endosymbionts with success. We submitted a book chapter on methods for detection and analysis of Wolbachia in PPNs. Our team presented at conferences. We successfully ran our High School Genomics Scholars and Bioinformatics Challenge programs. We integrated 3 undergraduates and 1 graduate as trainers for these programs. We accumulated of a large body of new data and protocols for the next experiments. Obj. 1.1 Analyze population genomic data from field-sampled PPN populations for signatures of selection on endosymbiont genes. (1) Activities: We focused on processing hundreds of samples collected in 2022 and performed additional nematode field sampling in 2023. We completed DNA isolation, and optimized a PCR screen to make our pipeline more cost-effective. We did Sanger sequencing and Illumina and PacBio sequencing on a subset of samples, followed by bioinformatic analysis to confirm results. We designed nematode primers for positive controls. (2) Data: We collected nematodes from 48 sites in 10 states in 2023. Analysis of the 497 collected nematode samples collected to date showed 193 samples to be positive for universal bacterial primers, 120 positive for Wolbachia (62.2%), 149 positive for Cardinium (79.7%), and 100 positive for both Wolbachia and Cardinium (53.5%). About 1/3 of sites were from farms with diverse crops. To characterize nematode genera/species, we performed 18S rRNA PCRs for amplicon sequencing. Illumina and PacBio sequencing on several samples showed the symbionts were at extremely low titer. Therefore, we performed dilution-PCRs to identify high titer samples for Illumina and PacBio sequencing. (3) Summary: Our data thus far suggest PPN endosymbionts are diverse and prevalent in farm and wild nematode communities. (4) Key outcomes: We submitted one book chapter and have presented these findings at various conferences. We trained 3 graduate students, 13 undergraduate students, and 8 high school students related to this work. Results provide a large biological resource of PPNs and endosymbionts. Obj. 1.2 Analyze drivers of evolution across global populations of PPNs and rhizosphere samples through bioinformatic database mining. (1) Activities: We developed python code for sequence database mining to look for endosymbionts in soil and rhizosphere databases. PhD student Era Sharma led these efforts and worked with 6 computer science undergraduates as part of the Bioinformatics Challenge summer program. (2) Data: We developed new software, called "SRAminer v2", which successfully processed hundreds of thousands of SRA files and found hidden Cardinium- and Wolbachia-like symbiont genomes in Globoderidae and Heteroderidae and soils. (4) Key outcomes: PhD Student Era, along with Bioinformatics Challenge mentees have polished and tested their data mining script, demonstrating its high efficiency and accuracy. MS student Taranjot completed and defended her Master's Thesis in 2023 on data mining to discover Wolbachia strain wHet and is developing a manuscript from this work. Obj. 2.1 Analyze Wolbachia and Cardinium endosymbiont effects on PPNs using root-cultures and measure fitness and microbe-host gene expression in these lines. (1) Activities: We improved methods to grow PPN lines for mono-symbiotic and aposymbiotic experiments. We modified our protocols and changed to pluronic media for convenient nutrient replacement and nematode extraction. Live P. penetrans were morphologically identified and picked from bulk nematode communities. (2) Data: P. penetrans were isolated from diverse localities with different symbiont status and placed in culture plates. (4) Key outcomes: These experiments are underway. Graduate students gained skills and developed improved protocols for generating reliable culture plates. Obj. 2.2 Assess endosymbiont effects on PPN parasitism on greenhouse plants and transparent soils. (1) Activities: Nematode communities from diverse P. penetrans populations (i.e., Wolbachia-positive, W-negative and Cardinium-positive, C-negative, and dual infected) have been inoculated into potted plants in the greenhouse for future studies. (4) Key outcomes: These experiments are underway. Obj. 3.1 Test endosymbiont effects on PPN parasitism in plant communities transplanted to the greenhouse under temperature stress. (1) Activities: We began testing of various field-collected PPN inocula in the greenhouse using different soils and watering regimes. We also performed tests of P. penetrans survival at different temperature treatments in vitro. (2) Data: In vitro tests over 3 days showed no change in survival at 32°C, ~10% mortality at 36°C, and ~70% mortality at 40°C. Results suggest our greenhouse are not ideal during summer/winter extremes. The greenhouse will undergo major renovations that may address this problem. (4) Key outcomes: These initial findings suggest practical ranges for further temperatures assays in vitro and in planta. Obj. 3.2 Develop and evaluate a computational model that probes endosymbiont-PPN-plant interactions under land-use and climate changes. (1) Activities: I advertised a postdoc position again this year, after failing to find any applicants in the first year who had suitably specialized skills and credentials. I reviewed incoming applicants and hope to find a suitable applicant this year to lead key aspects of this sub-objective. Edu Obj. 1: Develop a "Bioinformatics Challenge" Undergraduate Fellowship. (1) Activities: I developed and advertised the Bioinformatics Challenge Program through Texas Tech's associations for Black Students, Hispanic Students, and First-Gen Students, as well as the Math, Biochemistry, and Computer Science departments. Selected students signed a "best practices" contract, then were trained in HPCC and server/unix practices, meeting daily in small teams to work on bioinformatics code. (2) Key outcomes: Participants produced code and logged programming bugs and workarounds. We assessed effectiveness of training and compiled guides to improve our program for future years. Edu Obj. 2: Create a "Genomics Scholarship" peer-to-peer mentoring for high schoolers from underrepresented minorities. (1) Activities: PI Brown worked with local public schools to set up approvals for the program. We disseminated ads to attract diverse applicants and visited classrooms of 3 high schools to discuss the program. We interviewed and selected students and obtained trainings/permissions to work with minors. We trained Genomics Scholars in safety and and equipment use, then divided them into small research teams led by undergraduate and graduate mentors. (3) Summary: The program was successful in training 8 high school students from 3 local schools, fully involving them in the research objectives above. (4) Key outcomes: We received praise from students and local schools. We learned several aspects to improve for next year. Edu Obj. 3: Develop and pilot Rhizosphere Citizen Science and Rhizosphere Biomodelling Network programs. (1) Activities: Due to the large time investment and personnel investment in the two programs above, we postponed the major efforts in implementing our planned Rhizosphere Citizen Science program. I again advertised for a postdoc whose role will include helping to lead the Rhizosphere Biomodelling Network program.
Publications
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2023
Citation:
2023 Brown AMV. Widescale sampling and analysis of plant-parasitic nematode Wolbachia uncovers new strains and unsolved puzzles. 11th Wolbachia Conference, Jun 11-16, Crete, Greece.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2023
Citation:
2023 Kaur T (presenter), Brown AMV. Discovery of a novel Wolbachia strain associated with sugarbeet nematode suggestive of ancestral symbiosis. Texas Tech Annual Biology Student Symposium (TTABSS), Mar 31-Apr 1, Lubbock, TX.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2023
Citation:
2023 Kaur A (presenter), Brown AMV. Bioinformatics analyses reveals diverse genomes of Cardinium endosymbiont in soybean cyst nematode. Texas Tech Annual Biology Student Symposium (TTABSS), Mar 31-Apr 1, Lubbock, TX.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2023
Citation:
2023 Sharma E (presenter), Brown AMV. Analyzing hidden symbionts in sequence archives using a novel bioinformatic database mining software tool. American Society of Microbiologists (ASM) Microbe, Jun 15-19, Houston, TX.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2023
Citation:
2023 Kaur A (presenter), Brown AMV. Unearthing the hidden world: exploring the endosymbionts in nematode communities through soil sampling. Society of Nematologists Meeting, Jul 9-14, Columbus, OH.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2023
Citation:
2023 Kaur T (presenter), Brown AMV. Discovery of a novel Wolbachia strain associated with sugarbeet nematode suggestive of ancestral symbiosis. Texas Tech Graduate School Poster Competition, Mar 30, Lubbock, TX.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2023
Citation:
2023 Kaur A (presenter), Brown AMV. Comparative genomic analyses and enrichment test suggest evolutionary specialization of Cardinium symbiont. Plant and Animal Genomes Conference XXIX, Jan 13-18, San Diego, CA.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2023
Citation:
2023 Kaur T (presenter), Brown AMV. Discovery of putative Wolbachia HGT in the sugarbeet nematode suggestive of ancestral symbiosis. Plant and Animal Genomes Conference XXIX, Jan 13-18, San Diego, CA.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2022
Citation:
2022 Kaur A (presenter), Brown AMV. Bioinformatic analysis of Cardinium symbionts in soybean cyst nematodes. 61st Annual SON Conference, Sep 26-30, Anchorage, AK.
- Type:
Other
Status:
Published
Year Published:
2022
Citation:
2022 Brown AMV. Comparative genomics of endosymbionts Wolbachia, Cardinium, and Xiphinematobacter with potential to control plant-parasitic nematodes. Oct. 19, Department of Botany and Plant Pathology, Purdue University (Departmental Seminar), West Lafayette, IN.
- Type:
Other
Status:
Published
Year Published:
2022
Citation:
2022 Brown AMV. Paradoxically together: theory, discoveries, and new methods in analysis of emerging and long-term mutualisms. Sep. 21, Department of Biological Sciences, Texas Tech University (Departmental Seminar), Lubbock, TX.
- Type:
Theses/Dissertations
Status:
Published
Year Published:
2023
Citation:
2023 MS Thesis, Taranjot Kaur: Exploring the hidden tripartite association between bacterial endosymbionts, plant parasitic nematodes, and plants.
- Type:
Other
Status:
Published
Year Published:
2023
Citation:
2023 Brown AMV. CAREER: Discovering hidden drivers of rhizosphere symbiosis and parasitism. Feb. 15, National Science Foundation - National Institute of Food and Agriculture Plant Biotic Interactions Program Awardee Meeting (Virtual).
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Progress 09/01/21 to 08/31/22
Outputs
Target Audience:Target Audiences: The activities of the first year of the project reached numerous students primarily involved in the research (5 graduate students and 1 undergraduate student), approximately 81 other students at our institution through classroom presentations, and academics, professionals, and the public through dissemination activities (a conference poster, an invited departmental seminar, and a peer-reviewed publication with ~1,500 views and 263 downloads thus far). Students primarily trained and engaged in the research include 3 PhD students and 2 MS students (100% female, 1 Hispanic/Latinx, 3 South Asian) and one undergraduate. Students in the classrooms include diverse populations representing the demographic profile typical at TTU, which is a Hispanic Serving Institution with a large percentage of first-generation College students and students with rural backgrounds. Efforts: Efforts made include presentations on the methods and outcomes of this work in formal classroom situations (in PI Brown's class, Microbiomes), and in PI Brown's course based undergraduate research experience (CURE) course (Frontiers in Host-Microbe Genomics). Additional efforts were made to expose undergraduates and a first generation Hispanic non-thesis MS student (Amanda Ramirez) in project-related skills in the laboratory and in the field. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?Training Activities: This project year provided one-on-one training opportunities with a mentor for five graduate students and one undergraduate student who focused on this project. The first graduate student focused entirely on this project (Amandeep Kaur, PhD student) has been extensively trained in designing and tested a new PCR assay for detection of endosymbionts in bulk nematodes, and has been trained in bioinformatics (bash shell scripting, python coding, and using numerous other omics software tools), genomic library preparation, and nematology methods. The second graduate student focused entirely on this project (Taranjot Kaur, MS student) began in September 2021, and has been trained by the project in nematology, genomics, bioinformatics, and development of specific laboratory experiments on P. penetrans using the NemaLife physiological assay platform (in the engineering lab of Dr. Siva Vanapalli at TTU) and HPLC to isolate plant exudates (in the chemistry lab of Dr. Kristin Hutchins) to investigate the effects of root exudates on the nematodes. The third graduate student focused entirely on this project (Era Sharma, PhD student) began in January 2022, and has been training in bioinformatic software development (python, git, bash, C++, SQL, and specific programs) to build advanced pipelines for our work. The fourth graduate student (Shiva Abdollahi, PhD student) is primarily involved in a different project, but she has invested two semesters in this project, developing PPN-Wolbachia-specific primers and a PCR assay, doing field work to collect nematodes in Florida, and performing amplicon library preparations and metatranscriptomic bioinformatic analyses. She was also trained in preparation and publication of a peer-reviewed manuscript. The fifth graduate student (Amanda Ramirez, non-thesis MS student) was actively involved in leading two months of summer field sampling trips to collect approximately 200 nematode samples. The undergraduate student (Micah Garner) has worked for a full year on this project, becoming trained in a wide range of laboratory skills, best practices, data analysis, and command line bioinformatics. Professional Development: PI Brown was trained in advanced methods of pedagogy (applicable to this project) through enrollment and participation from September through May in the STEM Teaching, Engagement & Pedagogy (STEP) program at Texas Tech University. This program involved weekly team workshops on special topics in active learning, diversity, equity and inclusion in education, effective development of course based undergraduate experiences (CUREs), and effective alternatives. Through the STEP program's expert facilitators at Texas Tech's Teaching, Learning, and Professional Development Center, PI Brown also had her lectures assessed and critiqued with several sessions involving feedback from education professionals. PhD student Kaur gained professional development experience through travel to Oregon to work with and learn techniques in nematology from Dr. Inga Zasada. She also gained skills in presenting her work though developing and presenting a poster on her work related to this project at a national conference (Plant and Animal Genomes Conference XXIX, San Diego, CA. Note: at the last minute, the conference was converted to online format due to the rise in the Omicron variant of COVID-19). How have the results been disseminated to communities of interest?Some results from this work were presented to both graduate and undergraduate students (approximately 70 undergraduates and 11 graduates) with a broad range of degree of study majors or fields of interest at Texas Tech University, through presentations of work in progress by the PI and graduate students in the courses "Microbiomes" and "Frontiers in Host-Microbe Genomics". Some of the results of this work were presented to faculty and students with broad interests in crop sciences and agriculture at the Seminar of the Department of Plant Pathology at Kansas State University, through PI Brown's presentation as Invited Speaker on Dec. 9, 2021, with seminar titled "Comparative genomic insights into nematode symbioses." Several findings were presented as a poster presentation to broad audiences in plant and animal genomics by PhD student Amandeep Kaur at the Plant and Animal Genomes Conference XXIX, San Diego, CA (virtual due to Covid-19), Jan. 8-12, 2021. Some results of this work were disseminated in our 2022 peer-reviewed paper in the journal Frontiers in Microbiology (Journal Impact Factor 6.064), titled Discovery of early-branching Wolbachia reveals functional enrichment on horizontally transferred genes (doi.org/10.3389/fmicb.2022.867392). To date, this article has 263 downloads and 1,499 online views (570 in USA, 119 in China, 69 in Germany, 54 in Italy, 140 in other countries). What do you plan to do during the next reporting period to accomplish the goals?Now that the project has three graduate students (two PhD students and one MS student), all with experiments underway, we will work extensively with completing the objectives (1.1, 1.2, 2.1, 3.1) as per our timeline. From the extensive 2021-2022 field sampling across the U.S., we will continue pre-screening bulk nematode and picked nematode samples using the modified two-step PCR. We will also proceed with short-read and long-read metagenomic sequencing for all samples with positive results for Wolbachia and Cardinium, as well as sequencing several additional populations to search for additional low-level endosymbionts not detected in the PCR. We will continue with population genomics analyses on our high-quality circular endosymbiont genomes with our newly modified combined pipeline of metaWRAP (modified code) and an in-house python code we developed this year. Although initially, during the peak sampling season (summer/fall 2021) we were impacted by COVID-19, with some challenges with accessing some of our target sample sites, particularly farms, by summer of 2022, we were able to adequately resume the field work. Laboratory work with live P. penetrans was more challenging than expected in the first year of the project. The primary issue was contamination of the trial corn root agar after inoculation with isofemale starter populations. Greenhouse plants were also challenging to inoculate and maintain due to malfunction of venting during extreme heat of the summer and occasional malfunction of water supplies. The greenhouse is scheduled for and major upgrades that should alleviate these issues. We also plan to perform parallel experiments in departmental growth chambers. As we overcome the issues described above, we will expand our Gamborg's media agar root culture work over the summer, fall, and winter, and refine our antibiotic/heat clearing tests of nematode-endosymbiont populations on these plates. We will launch a new larger-scale replicated greenhouse population for our target nematodes (P. penetrans) and other suspected Wolbachia hosts such as Helicotylenchus spp., and several others that we have identified in our PCR pre-screening. We will re-sample only sites that had positive endosymbiont results, and begin our outreach (Citizen Science Program) to increase the cost-effectiveness of re-sampling at these sites. In parallel with aposymbiotic trials above, we will set up and assess our first temperature tests in native transplanted grasses and turfgrass collected from a university-associated research plot near campus which we found to have P. penetrans. Outreach and educational activities will be a major focus for the year ahead. Specifically, we will finalize our IRBs for the High School Genomics Scholars program which will launch next spring/summer, and we will begin to advertise and launch the summer tiered-mentoring Bioinformatics Challenge program for undergraduates. I will lead in building these programs, which will then, in future years integrate with the Rhizosphere Citizen Science and Rhizosphere Biomodelling Network. An additional goal in the year ahead is to re-advertise and hire a strong postdoc for our team. My fall 2021 postdoc ad resulted in a small pool of 14 applicants, whose applications were carefully reviewed, but none had satisfactory computational experience. Because of some of the advanced bioinformatic and modeling goals of this project, I will revise the ad to emphasize these skills and disseminate the ads more effectively and broadly, in order to have the best chance of attracting a postdoc who can contribute high-level research skills required for Obj 2 and 3.
Impacts
What was accomplished under these goals?
IMPACT: This project focuses on bacteria within nematodes infecting plants, causing losses to global crop yields estimated at $100 billion annually. The goal is to discover how the bacteria might be used to control these pests. The approach centers on advanced genomics combined with field and laboratory studies to determine the functions of the bacteria and how they impact nematode parasitism on plants under stress. Preliminary data suggests these bacteria could become targets for sustainable control of these extremely difficult to control nematodes. In the first year, PI Brown with 5 graduates and 1 undergraduate student, performed nematode field sampling from hundreds of sites across the U.S., screened populations, and found a rich diversity of potential endosymbionts. Experiments have begun to clarify activities of these symbionts in their nematode hosts. We published a peer-reviewed paper showing genes implicated in nutrient pathways and widespread presence of the endosymbionts. Other outcomes include a large biological resource, manuals and protocols for our upcoming educational and outreach goals, and advanced training of our team. Obj. 1: Determine the ecological and evolutionary forces acting on PPN endosymbiont function. Obj. 1.1 Analyze population genomic data from field-sampled PPN populations for signatures of selection on endosymbiont genes. (1) Activities: We performed extensive nematode field sampling. We began DNA isolation, sequencing, and bioinformatic analyses and established new local study sites with rich PPN biodiversity. (2) Data: We collected nematode communities from 410 sites across 23 states (TX, NM, OK, KS, OR, GA, FL, AL, TN, AR, SD, AZ, CA, WY, UT, NE, MI, ID, NV, MO, LA, MS, IA), with ~650,000 nematodes in hundreds of genera. Most samples had many PPNs; 5% of samples had >100 P. penetrans for live experiments in Obj. 2. A new two-step PCR was developed and tested, revealing half of tested samples had Wolbachia while most had Cardinium. Most P. penetrans samples from OR were doubly infected. Bulk nematodes revealedone quarter infected with Wolbachia and all infected with Cardinium. (3) Summary: We developed an efficient pipeline to collect and pre-screen a large diversity of PPNs for shotgun sequencing and population genomics. (4) Key outcomes: We published one paper on these findings describing a new PPN-type Wolbachia in Texas. We provided advanced training for 5 graduate students. Results also provide a large biological resource of PPNs and endosymbionts. Obj. 1.2 Analyze drivers of evolution across global populations of PPNs and rhizosphere samples through bioinformatic database mining. (1) Activities: We performed sequence database mining to look for endosymbionts in published PPN sequences and soil and rhizosphere sequences. We developed new efficient bash/python scripts for data mining and used these on our shotgun sequence data. (2) Data: We found a divergent, early-branching PPN Wolbachia with genomic features between mutualist and reproductive parasites. Functional analysis indicated enhanced heme, thiamine, and lysine biosynthesis and purifying selection on arginine and lysine metabolism, vitamin B6, heme, and zinc ion binding. A eukaryote-like gene that may mediate plant systemic acquired resistance through the lysine-to-pipecolic acid system. With our in-house data-mining code, we analyzed Wolbachia-like variants from global rhizosphere databases and uncovered dozens of putative PPN-type Wolbachia. We found a genome of Wolbachia in Heterodera schachtii. We uncovered Cardinium-like symbionts from PPN and rhizosphere data. We found 5 Heterodera glycines samples contained Cardinium genomes with functional divergence across populations and similar sequences in Globodera rostochiensis, Globodera tabacum, Heterodera avenae, and H. schachtii. (3) Summary: Results support plant-specialization and present new functional hypotheses on the role of these bacteria in these important PPNs. (4) Key outcomes: One paper was published. PhD student Amandeep has presented the Cardinium data-mining results at a conference and is currently working on manuscript from findings in this study. MS student Taranjot is developing a manuscript from this work. Obj. 2: Directly test PPN endosymbiont effects on host nematode fitness, phenotype, and parasitism on plants using symbiont-clearing experiments. Obj. 2.1 Analyze Wolbachia and Cardinium endosymbiont effects on PPNs using root-cultures and measure fitness and microbe-host gene expression in these lines. (1) Activities: We worked extensively toward improving methods to grow PPN iso-female lines for mono-symbiotic and aposymbiotic experiments. (2) Data: 3000 P. penetrans were isolated and 60 were inoculated into root agar plates using a modified seed sterilization protocol we developed. (3) Summary: Plate contamination and low reproductive rates under laboratory conditions is an ongoing challenge. (4) Key outcomes: Graduate students gained skills and developed improved protocols for generating reliable corn root plates. Obj. 2.2 Assess endosymbiont effects on PPN parasitism on greenhouse plants and transparent soils. (1) Activities: Preliminary work was performed, collecting and growing P. penetrans under various conditions. (4) Key outcomes: Preliminary skills-building in live PPN handling for team members. Obj. 3: Evaluate and model the dual effects of PPN endosymbionts and environmental stress on PPN-plant community interactions. Obj. 3.1 Test endosymbiont effects on PPN parasitism in plant communities transplanted to the greenhouse under temperature stress. (1) Activities: Testing field-collected PPN inocula in the greenhouse using different soils and watering regimes. (2) Data: Results suggested sandy soils were not suitable for P. penetrans from OR. Fall to spring conditions were more favorable. (4) Key outcomes: Preliminary tests provided information for future experiments. Obj. 3.2 Develop and evaluate a computational model that probes endosymbiont-PPN-plant interactions under land-use and climate changes. (1) Activities: I advertised a postdoc position and reviewed applications. The prospective postdoc would partially lead other objectives listed above and below. (4) Key outcomes: No postdoc applicants were deemed sufficiently trained for the position, so efforts were made to re-write the position based on these outcomes. Edu Obj. 1: Develop a "Bioinformatics Challenge" Undergraduate Fellowship. (1) Activities: We developed code, coding guides and strategic plans for the summer 2023 bioinformatics program. I gained expert pedagogy training through the TTU Pedagogy (STEP) program. I met weekly with mentors and colleagues to workshop improved teaching techniques. The STEP program also came to my classes to evaluate my teaching and provide detailed guidance to improve my teaching. (2) Data: Training guides for samsa2, metaWRAP, bwa/samtools, metaSPAdes, blastn/p, Diamond, QIIME2, bash scripting and python coding. Edu Obj. 2: Create a "Genomics Scholarship" peer-to-peer mentoring for high schoolers from underrepresented minorities. (1) Activities: We have begun to develop an IRB and outreach plan and we have begun training graduate students in mentoring of high school students. We re-worked and tested hands-on genomics protocols and manuals. (3) Summary: We continued working with High School Clark Scholars in 2021 and 2022 and will adapt protocols and approaches from this educational framework. (4) Key outcomes: Protocols and manuals for genomics aimed at introductory levels were developed for this program. Edu Obj. 3: Develop and pilot Rhizosphere Citizen Science and Rhizosphere Biomodelling Network programs. (1) Activities: We discussed and planned the Rhizosphere Citizen Science program, revising protocols based on preliminary screening results. I advertised for a postdoc, one of the duties of which will be to partially lead the Rhizosphere Biomodelling Network program.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2022
Citation:
2022 Weyandt N, Aghdam SA, Brown AMV. Discovery of early-branching Wolbachia reveals functional enrichment on horizontally transferred genes. Frontiers in Microbiology 13:867392. doi.org/10.3389/fmicb.2022.867392
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2022
Citation:
2022 Kaur A (presenter), Brown AMV. Metagenomic analysis of soybean cyst nematodes reveals diverse genomes of the Cardinium endosymbiont. Jan. 8-12, Plant and Animal Genomes Conference XXIX, San Diego, CA (virtual due to Covid-19).
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