Progress 09/01/18 to 08/31/23
Outputs
Target Audience: The main target audience of this project is other scientists: Weed scientists who work on Cuscuta and geneticists and molecular biologists who focus on plant pathology and small RNA regulatory functions. Our efforts have reached these constituenciesprimarily via scholarly publications and talks delivered at meetings, outlined elsewhere in this report. Changes/Problems:We anticipate at least one more major publication to derive from this project: the sequence and analysis of the two newCuscuta?genomes. What opportunities for training and professional development has the project provided?This project supported the training and professional development of multiple Ph.D. students and undergraduate researchers. These students were mentored in scientific research, data presentation, and communication skills. The project played an intergral role in the final Ph.D. theses of two students (Nata Johnson and Collin Hudzik), and also in the ongoing thesis research of several other Ph.D. students (Yachi Nien, Allison Vanek, and Juan Cerda). How have the results been disseminated to communities of interest?Via publications, websites, and open-access Ph.D. theses(as described in this final report and the previous annual reports). Also via lectures and seminars delivered by the PIs and project participants at various meetings. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
All goals for Aim 1 were accomplished and published inThe Plant Cellby Hudzik et al. (2023). Aim 2 was mostly accomplished during the project period, and included a significant expansion: High-quality genome assemblies were created for bothC. campestrisandC. gronovii. Annotation and publication are still ongoing and data will be public as soon as possbile in both publication form and on the web at https://plantsmallrnagenes.science.psu.edu/Studies/Cerda Aim 3 was partially completed. Specifically, it was determined thatC. campestris trans-species miRNAs are made byhost biogenesis factors. This was included in the Hudzik et al. (2023) publication. The other approaches originally outlined in Aim 3 were inconclusive based on techinical difficulties with the experiments.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2023
Citation:
Hudzik, Collin, Sean Maguire, Shengxi Guan, Jeremy Held, and Michael J Axtell. Trans- Species microRNA Loci in the Parasitic Plant Cuscuta Campestris Have a U6-like snRNA Promoter. The Plant Cell, March 10, 2023, koad076. https://doi.org/10.1093/plcell/koad076.
- Type:
Websites
Status:
Awaiting Publication
Year Published:
2023
Citation:
https://plantsmallrnagenes.science.psu.edu/Studies/Cerda
Website for sharing of newly assembled Cuscuta campestris and Cuscuta gronovii genomes and annotations. Currently under password protection pending publication.
- Type:
Theses/Dissertations
Status:
Published
Year Published:
2023
Citation:
Investigating interface-induced microRNA accumulation and regulation of the parasitic plant Cuscuta campestris
https://etda.libraries.psu.edu/catalog/23438cmh447
|
Progress 09/01/21 to 08/31/22
Outputs
Target Audience: The main target audience of this project is other scientists: Weed scientists who work on Cuscuta and geneticists and molecular biologists who focus on plant pathology and small RNA regulatory functions. Our efforts have reached these constituencies this year primarily via scholarly publications and talks delivered at meetings, outlined elsewhere in this report. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? During this period, the project has involved 4 Ph.D. students (Collin Hudzik, Allison Zvarick, Yachi Nien, and Juan Cerda), 1 postdoc (Imen Tammich), and 2 Technicians (Paula Ralph and Hope Gruszewski). Ph.D. student Collin Hudzik has completed his manuscript, posted it in bioRXiv, and submitted for peer review. All of the participants have benefitted from professional development in terms of regular lab meetings, presentations, and in the case of the grad students, major writing projects culminating in published papers and a dissertation. Allison Zvarick was able to attend this year's World Congress on Parasitic Plants in Kenya where she presented her research and made connections in the field. All four of the Ph.D. students participated in a meeting (The 23rd Penn State Symposium in Plant Biology: RNA Biology) that was organized by PI Axtell at Penn State in May 2022. The project also involved four undergraduate researchers. One, Miles Zakos, completed his honors thesis in May 2022 by studying the microRNAs of Cuscuta gronovii. How have the results been disseminated to communities of interest? Through scholarly publications and meeting presentations (poster and oral),as described elsewhere in this report. What do you plan to do during the next reporting period to accomplish the goals?The RNA-seq experiments will be completed, followed by annotation of the Cuscuta gronovii genome. Analysis of this genome will then commence, with a focus on the trans-species microRNAs and on genes potentially involved in Cuscuta virulence. The data will be published along with a useful website and genome browser interface. The newly designed microRNA target mimics will be tranformed into host plants, and then used for assays to directly assess the contributions of Cuscuta campestris trans-species microRNAs to virulence.
Impacts
What was accomplished under these goals?
Aim 1 experiments were completed and the results included in a preprint / manuscript that is now under peer review (see products). Aim 2 actually now focuses onCuscuta gronovii, notCuscuta campestris. This year, we have completed a chromosome level assembly of theCuscuta gronoviigenome.A detailed series of diverse RNA samples have been collected and are currently being used for mRNA-seq and microRNA-seq experiments. These experiments will allow an accurate annotation of theCuscuta gronoviigenome sequence.At the same time, we have generated a new assembly ofCuscuta campestrisusing long reads that is much better than the published genome and are working to resolve a chromosome level assembly. Aim 3 experiments have been re-designed and are ongoing. In particular the additional knowledge about Cuscuta microRNAs that we've gained has led us to what we think should be a more robust design for the miRNA-target mimic transgenes. These new and improved mimics have been generated and are currently being transformed into host plants for the definitive experiments. Additional details: Toward the goal of studying the effect of host miRNAs on Cuscuta gene regulation, we tested several methods of applying exogenous miRNAs directly to Cuscuta tissues. Although miRNAs were successfully infused into Cuscuta, we detected no impact on expression of their target mRNAs. From this we conclude that miRNAs require some biological processing or assistance to effectively silence genes in Cuscuta, as we have demonstrated that miRNAs produced in an attached host and translocated into the parasite are effective. To further describe the C. campestris - host interaction, we developed a synthetic promoter element that is specifically expressed in host tissue in response to Cuscuta parasitism. The promoter is responsive to wound stimuli associated with parasitism and presents a new tool for driving expression of genes in primarily in tissues being invaded by haustoria.
Publications
- Type:
Journal Articles
Status:
Under Review
Year Published:
2022
Citation:
Trans-species microRNA loci in the parasitic plant Cuscuta campestris have a U6-like snRNA promoter. Collin Hudzik, Sean Maguire, Shengxi Guan, Jeremy Held, Michael J. Axtell. bioRXiv (preprint) https://doi.org/10.1101/2022.07.06.498962
- Type:
Websites
Status:
Published
Year Published:
2022
Citation:
https://plantsmallrnagenes.science.psu.edu/Studies/Hudzik/
.. supporting data and data visualization for Hudzik et al. (2022) study.
- Type:
Journal Articles
Status:
Published
Year Published:
2022
Citation:
Westwood, J. 2022. Cracking open the witchs spell book: The witchweed genome provides clues to plant parasitism. New Phytologist. https://doi.org/10.1111/nph.18398
|
Progress 09/01/20 to 08/31/21
Outputs
Target Audience:The main target audience of this project is other scientists: Weed scientists who work on Cuscuta and geneticists and molecular biologists who focus on plant pathology and small RNA regulatory functions. Our efforts have reached these constituencies this year primarily via scholarly publications, outlined elsewhere in this report. Changes/Problems:The pandemic has continued to impact productivity. We have endured various and ever-changing restrictions of lab occupancy both at Penn State and Virginia Tech. This has impacted productivity. We are doing our best but obviously the course of the pandemic and the associated restrictions on lab occupancy are beyond our control. One change is on Aim 2, again. We have decided to not only sequence and assemble the ~1.9GbC. gronovii genome for the first time; we are also planning to bo back and re-sequence and assemble the ~0.5Gb C. campestris genome. This is because of the low cost and amazing quality of PacBio HiFi sequencing data. And also because the exisitng assemblies of the C. campestris genome are fragmented and low quality. Producing two high-quality reference genomes and annotations for the two most agriculturally relevant Cuscuta species will be a major outcome of this project. What opportunities for training and professional development has the project provided?During this period, the project has involved 4Ph.D. students (Collin Hudzik, Allison Zvarick, Yachi Nien, and Juan Cerda), 1 postdoc (Imen Tammich, and 3Technicians / Researchscientists (Paula Ralph, Eric Wafula, Matt Endres). Ph.D. student Collin Hudzik has single-handedly tackled aim 1, and has now wrapped up the experiments. He is writing the results up as a paper. All of the participants have benefitted from professional development in terms of regular lab meetings,presentations, and in the case of the grad students, major writing projects culminating in published papers and a dissertation.One of our normal professional development opportunities, summer meetings, unfortunately did not happen this summer AGAIN dueto the ongoing pandemic. We were however able to participate in Zoom-based seminars organized by the International Parasitic Plants Society (in lieu of their normally scheduled world congress, which has been postponed because of COVID). How have the results been disseminated to communities of interest?Through scholarly publications, as described elsewhere in this report. What do you plan to do during the next reporting period to accomplish the goals?Aim 2 : We are focusing our efforts on mRNA-seq and small RNA-seq from a rigorously defined set of tissues and treatments. These data will be used to annotated the C. gronovii genome assembly. We are also re-visiting the scope of aim 2 to once again include genome sequencing and assembly of C. campestris. This is justified because of the very low-cost of obtaining PacBio HiFi data coupled with the rather fragmented C. campestris genome assembly currently available; re-assembly of this species, the most agriculturally relevant of all Cuscuta species, is warranted and feasible. Aim 3: We are focusing on testing all of the various transgenic host plants that have been made for this aim. One major point of emphasis will be development of improved bio-assays that measure Cuscuta fitness / growth on hosts. This has been a major issue for this aim. More broadly, given that Cuscuta has evolved a system for manipulating host gene expression via trans-species small RNAs, it is relevant to also explore the effect of host miRNAs on the parasite. This phenomenon has also been raised as a potential vehicle for generating parasite-resistant crops. Relevant questions being explored include 1) Can Cuscuta gene expression be manipulated by exogenously supplied miRNAs? and 2) What is the best method for expression and delivery of miRNAs from the host? Answers to these questions will facilitate studies of Cuscuta biology.
Impacts
What was accomplished under these goals?
The experiments and analyses for Aim 1 were completed. These are moving toward publication very soon. We found that all trans-species MIRNAs accumulate very early in haustorial development .. in fact they appear before the haustorium penetrates the host, and they can also be coaxed to appear when haustorial development is stimulated in the absence of a host. There appears to be no host specificity whatsoever in the accumulation of Cuscuta trans-species microRNAs. As explained in previous reports, Aim 2 has shifted to genome assembly of Cuscuta gronovii. We have sequenced and assembled the ~ 1.9Gb C. gronovii genome to near chromosome level using PacBio HiFi data. Current efforts are directed toward annotation of this genome assembly using a combination of mRNA-seq, small RNA-seq, and computational predictions. Aim 3 has moved slower .. we have generated a lot of the required transgenic host plants, but a variety of factors have so far prevented us from obtaining definitive results. Aim 3 is a major focus for the coming project year.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2021
Citation:
Albert, Markus, Michael J Axtell, and Michael P Timko. Mechanisms of Resistance and Virulence in Parasitic PlantHost Interactions. Plant Physiology 185, no. 4 (April 23, 2021): 128291. https://doi.org/10.1093/plphys/kiaa064.
|
Progress 09/01/19 to 08/31/20
Outputs
Target Audience:The main target audience of this project is other scientists: Weed scientists who work onCuscutaand geneticists and molecular biologists who focus on plant pathology and small RNA regulatory functions. Our efforts have reached these constituencies this year primarily via scholarly publications, outlined elsewhere in this report. Changes/Problems:The biggest problem is the disruption caused by the COVID-19 pandemic. In-lab, on-campus research at both Penn State and Virginia Tech was largely shut down for a 3 month period (mid-March to mid-June 2020), and is now resuming at a reduced pace. This did slow down our work, although we were able to keep some activities going (data analysis and bioinformatics that could be performed from home). We are currently back in the labs, but at reduced capacity due to space / occupant density rules. We will continue to do our best in the face of the pandemic restrictions going forward. Another problem that arose was the departure of Jim Westwood's Ph.D. student, Chase Campanella. Chase decided to leave graduate school, which left Dr. Westwood's part of the project a bit stalled. Dr. Westwood will soon recruit a new postdoctoral associate for the project, but consequently there will be some delays in results. Because of both the pandemic and the personnel issue in Dr. Westwood's group, it is highly likely that we will request a no-cost extension next year in order to finish the project's major goals. That being said, the project has made very good progress, based on both publications and the as-yet unpublished data from aims 1 and 3. What opportunities for training and professional development has the project provided?During this period, the project has involved 4 undergraduate students,6Ph.D. students and2Technicians / Research scientists. The undergraduate students (Biology majors) benefitted from substantial research experience; this formed a key part of their studies. Ph.D. student Nathan Johnson completed his dissertation in late 2019 and earned his degree. Nate was a major contributor to this project, this is reflected in the publications to date, and he will be co-author on at least one more study related to this project. Ph.D. student Collin Hudzik has single-handedly tackled aim 1, and has made great gains in his studies this year. All of the participants have benefitted from professional development in terms of regular lab meeting presentations, and in the case of the grad students, major writing projects culminating in published papers and a dissertation. One of our normal professional development opportunities, summer meetings, unfortunately did not happen this summer due to the pandemic. How have the results been disseminated to communities of interest?Through scholarly publications, as described elsewhere in this report. The other typical method of dissemination, scientific meetings, were all cancelled this summer due to the pandemic. What do you plan to do during the next reporting period to accomplish the goals?Aim 1 is well in hand: We will be writing and submitting that manuscript early in the next reporting period. Aim 2 (genome sequencing ofC. gronovii) will be a priority. We will soon be able to make a decision on which isolate to use for genome sequencing and hopefully proceed rapidly with sequencing, assembly, and annotation after that. Aim 3 (functional analysis) has made good progress, but still requires a lot of work. In the coming period we will focus on the supporting molecular and genetic analyses required to shore up the conclusions we've made based on these experiments. We also need to begin the genetic reporter systems that were planned for this aim.
Impacts
What was accomplished under these goals?
During this reporting year, substantial progress was made on all three aims. Aim 1: Time-courses ofCuscutaattachment to two different hosts (Arabidopsis thalianaand tomato) have been completed and sRNA-seq data obtained. Analyses of these data are ongoing but preliminary results indicate no host specificity (the sametrans-speciesCuscutamiRNAs are induced on both hosts) and that all of thetrans-species miRNAs are induced early during the formation of haustoria (about 2 days after "coiling", which appears to be well before the host-parasite vascular connection is formed). We expect to submit a manuscript describing these results within the next 6-8 months. Aim 2: As described in last year's annual report, we have changed the genome sequencing focus fromC. campestristoC. gronovii. During this reporting year, we have collected specimens from multiple populations of wildC. gronoviiand we are currently performing experiments to estimate genome sizes. This is currently underway using Illumina skim-genome sequencing in order to generate k-mer based approximations of genome sizes. The isolate with the smallest genome will be selected for full sequencing and assembly. Final isolate selection and sequencing should occur in the autumn of 2020, with assembly and annotation being completed in early to mid 2021. Aim 3: The large number ofArabidopsis thalianatransgenic lines needed for this aim have been produced and characterized. During the past year we have been performingCuscutagrowth assays on these various lines. The results are very encouraging; we now have strong, direct evidence that selectCuscuta trans-species miRNAs directly affectCuscutagrowth. We have several follow-up experiments to conduct to firm up this conclusion for publication; these experiments will be conducted in the coming year.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2020
Citation:
Hudzik C, Hou Y, Ma W, Axtell MJ (2020) Exchange of small regulatory RNAs between plants and their pests. Plant Physiol 182: 5162
- Type:
Journal Articles
Status:
Published
Year Published:
2019
Citation:
Johnson NR, dePamphilis CW, Axtell MJ (2019) Compensatory sequence variation between trans-species small RNAs and their target sites. eLife 8: e49750
- Type:
Theses/Dissertations
Status:
Published
Year Published:
2020
Citation:
Johnson NR (2019) Function and maintenance of trans-species small RNAs in the Cuscuta genus of parasitic plants. Doctoral Dissertation, The Pennsylvania State University. https://etda.libraries.psu.edu/catalog/17261nrj113
|
Progress 09/01/18 to 08/31/19
Outputs
Target Audience:The main target audience of this project's efforts in the current year has been scientists: Weed scientists who work on Cuscuta, as well as geneticists and molecular biologists interested in plant pathology and small regulatory RNA functions. Our efforts have reached these constituencies in two ways: 1. Through scholarly publications (detailed elsewhere in this report) 2. Through presentations and interactions at a major international meeting (the World Congress on Parasitic Plants, held in early July 2019, in Amsterdam). The PI and both co-PIs gave presentations at this meeting. Participants from all continents (save Antarctica of course!) were present. Changes/Problems:Publications and progress by other scientists working in this general area have necessitated a change in Objective 2 (which was originally conceived as imrovement of the C. campestris genome assembly). A group from Norway (Vogel et al., 2018) published a pretty high-quality genome assembly and annotation of C. campestris, so our efforts along those lines would be moot. Our new plan is to generate a draft genome assembly and annotation for another species of Cuscuta, Cuscuta gronovii. C. gronovii is very common in the US and, like C. campestris, is a noxious weed that affects agriculture, in particular cranberry production in the North-Eastern US. We are now in the process of performing genome-size estimations for several C. gronovii isolates to determine the most feasible one to sequence. We will also change our sequencing strategy: Originally we proposed to use Pac Bio sequencing. In the meantime, we've piloted the use of Oxford NanoPore Techonology's (ONT) MinION device for genome sequencing of C. gronovii; the results were very encouraging. With just two flow cells (an investment of $1,000), we recovered about 16Gb of long reads (read N50 ~ 25kb). From these data we were able to make a preliminary assembly of this genome which includes about 95% of the gene space (estimated by comparison with the C. campestris assembly). We need more depth to make this a better assembly, but ONT-sequencing seems very feasible and cost-effective. Overall this change is a good one: The result will be a genome sequence for another major Cuscuta species that is a noxious weed in the US; this will empower not only our specific research into the microRNAs, but a wide number of other research programs. Citation: Vogel, Alexander, Rainer Schwacke, Alisandra K Denton, Björn Usadel, Julien Hollmann, Karsten Fischer, Anthony Bolger, et al. "Footprints of Parasitism in the Genome of the Parasitic Flowering Plant Cuscuta Campestris." Nature Communications 9, no. 1 (June 2018): 2515. https://doi.org/10.1038/s41467-018-04344-z. What opportunities for training and professional development has the project provided?This project has supported two Ph.D. students, Nathan Johnson and Collin Hudzik. Nathan is finishing his dissertation this autumn and is the lead author on a study currently under review. Collin is a 3rd year Ph.D. student working on parts of Objectives 1 and 3. In terms of their professional development, the project has allowed both of them to present their work at two meetings over the last year: The Mid-Atlantic ASPB meeting (in May in Maryland), and at the World Congress on Parasitic Plants in Amsterdam this July. The project has also provided research opportunities to three PSU undergraduates in the past year: Tanner Fell, Sarah Weston, and Noah Robertson. All of them have performed substantial research in support of the project, and either have already or will in the next year, present their research in poster form at the Penn State Undergraduate Research Exhibition. Itsuhiro Ko, a Penn State student in the Department of Plant Sciences, applied for and obtained summer REU support to study horizontal gene transfer in Cuscuta. At Virginia Tech, Ph.D. student Chase Campanella was recruited to work on this project. He has only been on the job for a few months, but is gaining opportunities for research. How have the results been disseminated to communities of interest?Results have been disseminated by scholarly publications (detailed in the Products section of this report) and by presentations at national and international meetings (Mid-Atlantic ASPB meeting, May 2019, University of Maryland and World Congress on Parasitic Plants, July 2019, Amsterdam). What do you plan to do during the next reporting period to accomplish the goals?In the coming year, most of Objective 1 should be completed. The revised genome sequencing plan for Objective 2 will be pursued and our goal is to have a draft assembly in hand next year. Objective 3 will be a main focus, because those experiments are more laborious and time-consuming, and the most interesting. So far the project is overall going well; papers are being published and there is a lot of exciting new findings coming from this system.
Impacts
What was accomplished under these goals?
Objective 1: We focused a lot on the host-specificity goal, and expanded it to include studies of parasite specificity as well. Small RNA profiling from seven different Cuscuta isolates (representing four different species), revealed that trans-species microRNA activity is common among different Cuscuta species. Most interestingly, we found that the trans-species microRNAs from various Cuscuta species were often different in sequence, but could be grouped into "superfamilies". Superfamilies often varied in a three nucleotide repeating pattern; these variations corresponded to synonymous site variation in host mRNA target sites. This is a unique finding and is the first concrete evidence in any system of strong selection pressure acting on trans-species small RNAs. The manuscript is under review ( a preprint is available athttps://doi.org/10.1101/675900). Objective 2: The goals of this objective have changed (see the Changes / Problems section of this report). Nonetheless, we've made significant progress. Besides the pilot sequencing described in Changes / Problems, we've also examined the genome of C. campstresis and improved it by making a detailed study of horizontal gene transfer events. Among many interesting findings, we observed that regions of the genome derived from horizontal gene transfer were enriched as small RNA sources. This suggests a novel hypothesis as to how trans-species microRNAs may be born. This was published in Nature Plants recently (https://doi.org/10.1038/s41477-019-0458-0). Objective 3: This objective requires the creation of a large number of transgenic plant lines. During this reporting year we focused on the required cloning, transformations, and characterizations. Most of the required transgenic lines are now in place and testing will commence during the coming year.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2019
Citation:
Clarke, Christopher R., Michael P. Timko, John I. Yoder, Michael J. Axtell, and James H. Westwood. Molecular Dialog Between Parasitic Plants and Their Hosts. Annual Review of Phytopathology 57, no. 1 (2019): null. https://doi.org/10.1146/annurev-phyto-082718-100043.
- Type:
Journal Articles
Status:
Published
Year Published:
2019
Citation:
Johnson, Nathan R, and Michael J Axtell. Small RNA Warfare: Exploring Origins and Function of Trans-Species MicroRNAs from the Parasitic Plant Cuscuta. Current Opinion in Plant Biology 50 (August 1, 2019): 7681. https://doi.org/10.1016/j.pbi.2019.03.014.
- Type:
Journal Articles
Status:
Published
Year Published:
2019
Citation:
Yang, Zhenzhen, Eric K. Wafula, Gunjune Kim, Saima Shahid, Joel R. McNeal, Paula E. Ralph, Prakash R. Timilsena, et al. Convergent Horizontal Gene Transfer and Cross-Talk of Mobile Nucleic Acids in Parasitic Plants. Nature Plants, July 22, 2019, 1. https://doi.org/10.1038/s41477-019-0458-0.
|
|