Progress 12/15/21 to 12/14/24
Outputs
Target Audience:The target audience reached includesscientists in both industry and academiawithin the fields ofmolecularplant-pathogeninteractions, plant immunity, and nematology fields.Throughout the duration of this project, I attended international meetings hosted by the American Phytopathological Society and International Society of Molecular Plant-Microbe Interactions. At these meetings, I delivered poster presentations to communicate my research findings funded by this fellowship. My poster presentations attracted scientists with various backgrounds and at different stages of their career. In addition to presenting my research at these meetings, I reached by target audience by attending workshops and events with industry scientists. These activities and opportunities to connect with my target audience allowed me to develop my communication skills, engage in career development, and prepare myself for a career post-graduation. Attendance at these meetings also provided the opportunity to interact with other graduate students within my field work and expand my science network. My research funded through this fellowship was further communicated via publication in the journal of Molecular-Plant Microbe Interactions. My first-author manuscript was selected as "Editor's Pick" and featured in an American Phytopathology news report. By publishing in Molecular-Plant Microbe Interactions, I was able to reach my target audience while further engaging with my professional societies. This fellowshipalso includedan outreachcomponent. Throughout the fellowship period,I mentored undergraduates, post-bachelorettes, and a high school student inthe laboratory. In addition to my mentorship efforts, Iwas active inoutreach eventsfacilitatedby Dr. Innes.Eventsincluded leading plant specific demonstrations forhigh schoolstudentsthat arepart of the RISE program at Indiana University (IU), volunteering for plant-based lessons at IU's Science Fest, and leading plant science experiments in 3rdgrade classrooms.My outreach initiatives throughout this fellowship period gave me the opportunity to communicate the importance of plant science which reached the general public. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?Throughout the project period, I was engaged in opportunities for training and professional development which enabled me to successfully defend my PhD, execute goals of this project, and prepare for a career post-graduation. At Indiana University, Roger Innes was my primary mentor throughout this project period. We met regularly to discuss the development of this project and resulting manuscript. Roger also helped me further development my writing skills through providing me with feedback during the preparation of this fellowship, annual reports, and mauscrupt/thesis writing. As my primary mentor, Roger provided me with opportunities to mentor undergraduate students interested in research. Through these opportunities, students worked closely with me on aspects of this project funded by the USDA. Working on this project exposed the trainees to problems in agriculture, molecular nematology, plant-pathogen interactions, and general molecular/lab techniques. The students I have trained have continued their science careers as research techs, medical students, and students in the sciences. In addition to the training opportunities my project offered, I became a stronger leader and communicator due to mentoring which will be pivotal in my career going forward. In addition to mentorship, this fellowship enabled me to travel to Dr. Thomas Baum's lab at Iowa State University. For one month, I worked alongside the Baum lab where I was trained in molecular nematology, nematode infection assays, and dsRNA synthesis for gene silencing. This training opportunity was one of my major goals outlined in this fellowship as Indiana University did not have these resources despite the relevance to my overall project. This training period made me more competitive for my post-doctoral position in industry, where I am continuing to work towards the development of new SCN prevention methods. In additional to the technical skills I gained during my visit, Thomas was an exceptional mentor and host. During my visit, Thomas helped me connect with nematologists in industry as I was preparing to finish my PhD. He helped arrange several meetings with these scientists, including one in which I got to visit Corteva for a tour of their facilities. Throughout the duration of the project, I participated in professional development events with the American Phytopathological Society. Each year at the annual meetings, I would seek out opportunities to learn more about careers in industry, develop interviewing skills, and network with industry scientists. These initiatives helped me be competitive for positions post-graduation and transition to a non-academic setting. How have the results been disseminated to communities of interest?The results of this research have been communicated to the Plant Health research community throughout the project period through poster presentations and networking at the International Society of Molecular Plant Microbe Interactions and American Phytopathological meetings. In addition, I gave a departmental talk at Indiana University and a talk at my program's research retreat. These opportunities allowed me to reach a broader science audience outside of my field. In the last reporting period, and throughout the project, I have engaged in outreach initiatives to spread awareness of plant pathogens, nematology, and how plant diseases impact food security. Engaging in these events allows me to talk about preventative strategies and address misconceptions about genome engineering in plants to people of all backgrounds and ages. This is relevant to the USDA, and my research interests, as there is new technology being developed for agriculture practice and it's important for the public to understand our food supply and efforts to ensure food security. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
The primary focus during the last funding period of this fellowship was to identify and characterize the interactions between SCN effectors and their plant targets. Due to the data collected on Heygly22189 (CPR1), I prioritized this effector for further characterization. Since CPR1 is a cysteine protease, I further characterized the interaction between CPR1 and its confirmed target in soybean (GmBCAT1) that was identified at the end of the last reporting period. Using transient expression assays inN. benthamiana,I showed that when GmBCAT1 is co-expressed with CPR1, there is a reduction in GmBCAT1 accumulation which suggests cleavage. Inactivation of CPR1 enabled GmBCAT1 to accumulate to similar amounts of the empty vector control, further suggesting cleavage activity. I proposed a model in which CPR1 targets GmBCAT1 for cleavage to inhibit function. To further support these results, I performed confocal microscopy of both CPR1 and GmBCAT1 to show the interaction likely occurs in the mitochondria. Since mass spectrometry data from miniTurbo experiments yield 2-15 high-priority targets per effector, I prioritized further investigation of Hetgly13995 and Hetgly10202 targets. These effectors harbored domains present in other plant-parasitic nematode effectors that function during infection. During this reporting period, I generated a second mass spectrometry dataset to improve upon the first trial. This dataset was analyzed to determine a list of top priority candidates for each effector. To ensure to longevity of the data and future analysis, these datasets were shared with both the Baum and Innes labs for further investigation. The datasets for Hetgly14678 and Hetgly15757 were distributed in the same manner. In addition to data collection and sharing for future research, I did a literature review of the potential interactions between the effectors and putative plant targets from miniTurbo which is a chapter in my dissertation. The chapter describes similar effectors and/or proteins to my candidates in effort to predict function, the function of the plant targets, and how the characteristics of the effector/plant target could be associated to SCN infection. To further assess the function of CPR1, Hetgly13995, and Hetgly10202, I visited the Baum lab to perform nematode-based experiments and gain molecular nematology skills/techniques that are not available at Indiana University. The primary purpose of this visit was to collect nematode data to further support the CPR1 characterization for publication in a peer reviewed journal. While my original plan was to knock-out the targets of SCN effectors and then perform nematode infection assays, I focused on manipulation of the effector inside the nematode and inside the plant cell due to the valuable information it would provide and the time consideration given fall defense and graduation. In the Baum lab, I generated dsRNA to silence the CPR1 transcript. The CPR1-silenced worms were used in penetration assays which showed knock-down of CPR1 results in reduced penetration of juvenile nematodes into soybean roots, suggesting it to be a critical effector during infection. I generated constructs to express the effector candidates in composite soybean roots for nematode infection assays. From these experiments, I found plants expressing CPR1 were more susceptible to SCN infection than those expressing the control vector, further validating its importance during infection. This assay was carried out for Hetgly10202 withno statistical significance. Under my Training and Career Development objectives, I was engaged in these initiatives throughout the fellowship period. I attended a scientific conference each summer, apart from 2024 (due to thesis writing), to present my research findings and engage with my scientific communities and colleagues. In 2023, I attended both the American Phytopathological Society Meeting (Denver, CO) and Molecular-Plant Microbe Interactions (Providence, RI) where I presented my research and attended multiple networking events/workshops. I mentored 2 undergraduate students and a high school student during this fellowship. Each student had the opportunity to learn about the project funded by this fellowship, general molecular biology and plant techniques, and present their findings alongside me during lab meetings. These students have continued to have successful careers and further education in scientific fields. In addition to mentorship in the lab, I was engaged in outreach initiatives within the Bloomington community and state of IN. In 2023, I attended the USDA-facilitated PD meeting where I was able to connect and brainstorm project management strategies with fellow USDA predoctoral fellowship recipients. This day-long meeting was held on zoom and included break-out rooms to better communicate with attendees. In February 2024 I traveled to Dr. Baum's lab at Iowa State University where I stayed for one-month as part of this fellowship. During that month, I worked alongside a post-doc and research scientist to learn molecular nematology, infection assays, and dsRNA synthesis. My time during the Baum lab visit allowed me to complete two experiments for my manuscript describing CPR1 and further added to my skillset. In 2024, I prepared my first-author manuscript describing CPR1, which was accepted in the journal of Molecular Plant-Microbe Interactions. I also finished writing my thesis, which included the research funded by this fellowship, and defended my thesis to successfully graduate with my PhD. I successfully defended my thesis and submitted my dissertation in Fall 2024. My thesis is published on ProQuest and includes my published manuscript.
Publications
- Type:
Peer Reviewed Journal Articles
Status:
Accepted
Year Published:
2024
Citation:
Margets, A., Foster, J., Kumar, A., Maier, T. R., Masonbrink, R., Mejias, J., Baum, T. J.,
and Innes, R. W. 2024. The soybean cyst nematode effector Cysteine Protease 1
(CPR1) targets a mitochondrial soybean branched-chain amino acid
aminotransferase (GmBCAT1). Mol. Plant-Microbe
Interact.https://doi.org/10.1094/mpmi-06-24-0068-r.
- Type:
Theses/Dissertations
Status:
Accepted
Year Published:
2024
Citation:
Characterization of Soybean Cyst Nematode Proteases to
Engineer Durable Resistance in Soybean
Published on ProQuest
|
Progress 12/15/22 to 12/14/23
Outputs
Target Audience:The target audience reached during this reporting period includescientists in both industry and academia that are part of the plant-pathogen interactions, plant immunity, and nematology fields.My efforts to reach my target audience included attendance at the 2023 International Society of Plant Microbe Interactions meeting in Providence, RI and the 2023 American Phytopathological Society annual meeting in Denver, CO. At these conferences, I presented two different posters that highlighted research findings from this fellowship. My poster presentations welcomed scientists of various science backgrounds and career stages. In additional to presenting my research, I was able to reach my target audience by attending workshops and events with scientists within industry. These activities to connect with my target audience allowed me to further communicate my research and engage in career development. Attendance at these meetings also gave me the opportunity to touch base with othergraduate studentswithin my field who work on similar research questions as I do. Specifically, last reporting period I discussed that I was able to help a graduate student from another lab optimize a technique I am using as part of this fellowship to address their own research questions revolving around soybean cyst nematode (SCN). At the meeting, we shared our research progress over the past year using the technique. As this fellowship also includes an outreach component, I have continued mentoringundergraduatesin the laboratory. This past reporting period, a senior pre-med student joined our lab and is under my mentorship to gain research experiences. While their future goals are outside of plant biology, this student has been fully engaged in the research process, significance of SCN in agriculture, and how the molecular questions I have proposed in this fellowship can contribute to developing solutions to the pathogenic threat. In addition to my ongoing mentorship efforts to include undergraduates, I also partake in the outreach events facilitated by Dr. Innes. During this past reporting period, this included leading plant specific demonstrations forhigh school studentsthat are part of the RISE program at Indiana University (IU). Students get the opportunity to spend several weeks a summer on IU's campus to visit various labs and explore the different science disciplines the university offers as they prepare for graduate and college. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?As this project is part of the USDA-NIFA-AFRI Education and Workforce Development program, I (the PD) have included a "Training/Career Development Plan" in the project proposal. While themajor goal of the Training/Career Development Planwas to gain the experience and skillset needed to pursue a tenure-track faculty position in academia, my career track has shifted to also consider careers in non-academic settings (industry/government). My interest to potentially pursue a career outside of academia has not changed my overall training and career development objectives. During this past reporting period I have presented aspects of this project at the International Society of Molecular Plant Microbe Interactions (IS-MPMI) and the American Phytopathological Society (APS) meetings. At APS, I attended a workshop hosted by Bayer to learn about the hiring and interviewing process in industry. This workshop included a mock interview with customized feedback. This experienceallowed me tolearn harness my experiences throughout my training thusfar totaught me how to draw on my graduate training to answer behavioral based questions. I also attended a graduate student mixer with industry representatives,which led to further discussion on my project and interest in a future seminar about my research for the company. I also had the opportunity to attend the USDA-facilitated PD meeting via zoom during this past reporting period. This opportunity allowed me to connect with other predoctoral fellowship recipients and share our experiences, successes, and obstacles throughout the funding period. There were several opportunities to share advice on how to maximize our opportunities and productivity as a predoctoral fellow. Through this opportunity, I additionally expanded my network to include predoctoral fellows from a variety of differentUniversitiesuniversities and fields. Outside of meeting settings, I have also engaged in annual outreach initiatives at Indiana University. The opportunities during this funding period have included delivering lessons and leading plant-based experiments in 3rdgrade classrooms, partaking in IU's annual Science Fest, and leading demonstrations for high school students in IU's RISE program. Through these experiences, I have learned how to adapt to different audiences to effectively communicate important and engaging science information,which will continue to bea pivatolan import role of mine as a scientist regardless of the career direction I pursue next. At the end of the funding period, I met with Dr. Baum and his lab members at Iowa State University to finalize plans to travel to Iowa for training in nematology-based techniques that will directly contribute to my project and overall skillset. I had the opportunity to lead discussion on the specific experiments that I want to accomplish during my visit and how to prioritize the SCN effectors that I am pursuing. Since the end of the funding period, and before submission of this progress report, I have traveled to Dr. Baum's lab to perform nematode-based assays on three of the effector protein candidates. Throughout the month, I worked closely with a post-doctoral researcher (Anil Kumar) and a research scientist (Tom Maier). In addition to learning more about the biology of SCN and overall nematology community, I learned new techniques that I can apply to either the nematode or other systems (RT-qPCR,in situhybridization, dsRNA probe synthesis, and silencing experiments). Additionally, Dr. Baum helped me maximize my time in Iowa by introducing me to scientists within his network. I met with a former PhD student (Peter Howe) of Dr. Baum who is now the leader of an international genotyping team at Corteva. During this visit, I toured the Corteva campus, learned about the large-scale approach industry takes, and discussed careers in industry. Additionally, I had the opportunity to meet Dr. Tylka, a leader in nematology and extension scientist, where we discussed my project, our research interests, and my career goals. Dr. Tylka also helped me expand my network by sharing contacts for industry scientists working in molecular nematology. In just a month at ISU, I not only gained a deeper understanding of nematology, new skillsets, and an expanded network, but I grew immensely as an independent scientist. The interactions and experiences I had while visiting and working in Dr. Baum's lab were invaluable and have helped shape how I approach science. How have the results been disseminated to communities of interest?The direct results of this research have been communicated to the Plant Health research community through my poster presentation and networking at the International Society of Molecular Plant Microbe Interactions and American Phytopathological meetings that have been described several times throughout this report. In addition to these venues for communicating my research, I was invited to give a talk for Dr. Helm's lab (a USDA research scientist) at Purdue University. Like last year, I participated in Indiana University's annual Science Fest, where I had the opportunity to volunteer my time at the Innes lab booth "Plants Get Sick Too." Here I discussed in broad terms what I do as a plant biologist, the types of problems that exist in agriculture, how the Innes lab, and myself, andaddress these problems through research. I also highlight the soybean cyst nematode and the impact it has on agriculture and food security. Communication during outreach initiatives is especially important as it increases the awareness of plant pathogens, nematology, and the overall impact plant disease can have on food security. It also provides an opportunity to discuss preventative strategies and address misconceptions about genome engineering in plants. This is relevant to my research as my work focuses on developing a foundation required for developing pathogen prevention strategies.? What do you plan to do during the next reporting period to accomplish the goals?Under Objective 1,I will determine if Hetgly22189 is able to suppress other forms of ETI.I will be using a combination of bacterial effector proteins and a BAX system which are known to elicit cell death when expressed inNicotiana benthamianaleaves. Initial experiments will determine optimal optical density of each ETI-elicitator and then proceed with cell death assays and electrolyte leakage assays. These findings will finalize my immune suppression figure for a manuscript that describes the characterization of Hetgly22189. Under Objective 2, I willgenerate a mass spec dataset with putative interactors for Hetgly20453, further confirm the interaction between Hetgly22189, and test interactions for my other candidates of interest using my already generated list of putative targets.The cloning and testing of these targets is currently ongoing. In addition, I will further confirm the identified interaction of Hetgly22189 through co-localization experiments and aim to identify the amino acids of the target that are cleaved by the nematode effector. While Hetgly13995 and Hetgly10202 do not have immune suppression activity, I will continue to pursue these two effectors as they have domains that have become more present in cyst nematode effector biology but have not yet been characterized. The findings of experiments revolving around these two effectors will be organized for publication. To aid in the interpretation of research findings, I will be in close communication with Dr. Baum's group at Iowa State University and continue to meet with Dr. Innes regularly. To further characterize the targets of interest,I am traveling to Dr. Baum's lab at Iowa State University in February 2024. I will spend the month learning alongside Dr. Baum's lab members where I will generate composite plants expressing my effectors of interest for nematode infection assays. During this time, I will also learn how to knockdown effectors for functional analysis by soaking the nematodes in dsRNA. In addition to gaining nematology techniques, I will also learn how to design dsRNA probes, performin situhybridization, and execute RT-qPCR experiments. During the last year of the fellowship,I will organize all data into manuscripts and/or my dissertation to communicate the research findings.Research findings that are not complete for manuscript submission will be organized and shared with Dr. Innes and Dr. Baum.I will present my research findings on this project one last time in a conference setting at the American Phytopathological Society in Summer 2024.This conference will be useful for not only presenting my research but for networking for for my next stage in my career. I will be most interested in talking to people in industry or laboratories focused on nematology and/or plant immunity for a post-doctoral position. In fall of 2024,I will defend my thesis and complete my PhD. Together, these activities to promote research progress and career development will directly aid in the completing thegoalsof this fellowship.
Impacts
What was accomplished under these goals?
Under Objective 1, todetermine whether soybean cyst nematode (SCN) effector proteins function in suppressing plant immune responses, I have finished screening all effector candidates of interest for their ability to suppress plant immune responses. My original list of effector proteins I selected for this project included 11 different effectors. I have reduced this list to seven effector proteins based upon preliminary data and ability to clone and expressin planta. Of the seven effectors, I have shown that three are able to suppress plant immune responses. All seven were screened for effector-triggered immune suppression (ETI) and pattern-triggered immune suppression (PTI). However, no additional candidates from last reporting period were found to have immune suppression capabilities. The one candidate that was shown to suppress ETI, Hetgly22189, was prioritized as a candidate for further characterization. Due to its known characterization as a cysteine protease and presence of an inhibitor domain, I cloned additional variants of the effector to generate a catalytically inactive mutant and a processed form of the effector lacking the inhibitor domain. Upon including these variants in my ETI suppression assay, I showed that Hetgly22189 ETI suppression is dependent on its protease activity and the mature variant is the most sufficient at suppressing ETI-dependent cell death. These conclusions were made using a combination of qualitative cell death assays inNicotiana benthamianaleaves and quantitative electrolyte leakage assays to measure the cell death observed. Based upon the overall analysis of results that have stemmed from Objective 1, I prioritized the three immune suppressing effectors for miniTurbo. Of the effectors that do not suppress plant immune responses, I have selected Hetgly13995 and Hetgly10202 for further characterization due to their unique domains that are not yet understood in the plant parasitic nematology field. Thekey outcomesof the activities, data collected, and analysis of results under Objective 1 have included the completion of screening the selected SCN effector candidates for immune suppression, additional analysis of how different variants of Hetgly6633 (inactive/mature/full length) impact its ETI suppression capabilities, and a finalized list of effector candidates that I will pursue for further characterization under Objective 2. Under Objective 2, toidentify the soybean proteins targeted by soybean cyst nematode effectors and assess how knockout of these targets influence the phenotypes associated with infection, I have performed miniTurbo in composite soybean roots for five different effectors of interest (3 immune suppressors; Hetgly22189, Hetgly14678, and Hetgly15757. 2 non-immune suppressors with unique uncharacterized domains; Hetgly13995 and Hetgly10202) and generated mass spectrometry datasets of putative targets. I have also generated three YFP:miniTurbo datasets to aid in eliminating the proteins non-specifically biotinylated. Of the effectors that I have produced mass spec data for, I have been primarily focused on the ETI-suppressor, Hetgly22189. In brief, I selected those proteins that were exclusive to the Hetgly22189 dataset (and absent in other effector datasets), were significantly represented in the data, and/or had relevant links to SCN infection and/or plant immunity. These targets were cloned from cDNA into expression vectors to determine which are true interactions of Hetgly22189. Using co-immunoprecipitations (coIPs), I showed that Hetgly22189 coIPs with a branched chain aminotransferase (BCAT) that localizes to the mitochondria. Literature suggests this protein is involved in the first step of branched chain amino acid degradation. Since Hetgly22189 is a protease, I performed cleavage assays to determine if the BCAT is cleavable by the effector protease. In brief, I co-expressed the active, inactive, or mature active Hetgly22189, variant with the BCAT and performed immunoblotting to detect the abundance and putative cleavage product of the BCAT when co-expressed with the active Hetgly22189 variants. While there was no cleavage product detected, the immunoblot analysis showed a significant reduction in full length K7KT64 when co-expressed with the active protease variants, but not the inactive variant. The coIP and cleavage test experiments were repeated 3 times with consistent results. Thekey outcomesof the activities, data collected, and analysis of results under Objective 2 have included the generation of mass spec datasets for 5 of the SCN effector proteins of interest each of which have yielded a list of top priority candidates to test to confirm interactions. Additionally, these datasets collectively have allowed me to identify and confirm a true plant target of Hetgly22189 which has been the top priority effector to characterize from the start of this fellowship.Training and career development objectives are described in the next section of this report.?
Publications
|
Progress 12/15/21 to 12/14/22
Outputs
Target Audience:The main target audience reached by my efforts during this reporting period includedscientists within the field of plant-pathogen interactions, plant immunity, and nematology.My efforts included attendance at the 2022 American Phytopathological Society annual meeting in Pittsburgh, PA. Here, I was able to present my research to scientists that were part of academia, industry, and government. The main effort to reach my target audience included a poster presentation during the conference. Other efforts included participation at a graduate-industry networking lunch and networking with other attendees at various levels within their career. Specifically, my target audience included severalgraduate studentswho visited my poster. After the meeting concluded, I was able to help one of the graduate students optimize a technique that I am using for this research project so they could utilize the protocol for their own research questions involving the soybean cyst nematode. My mentoring opportunities this reporting period were targeted to a localhigh school studentwho is currently in her junior year. My efforts included providing an overview of my research goals under this fellowship, sharing relevant literature, and teaching basic laboratory skills so she could assist in performing experiments such as cloning, transient expression assays, and bacterial/plant maintenance. This fellowship also includes an outreach component. During this reporting period, my target audience that was reached with my efforts included3rdgrade studentsin Bloomington, IN andpeople, of various ages and backgrounds, from throughout Indiana. My efforts with the 3rdgrade students included teaching the scientific process where students designed and performed an experiment focused on what plants need to grow. The students collected data and formed conclusions about the role light plays in plant growth. This effort allows me to broadly take my experience as a scientist and communicate how science is performed. Additionally, my efforts with the Indiana community included participation in Indiana University's annual science fest with the Innes lab at the "Plants Get Sick Too Booth." Here, I discussed broadly about plant-pathogens, agriculture practices, efforts by scientists to secure our food supply, and briefly on my work included in this USDA fellowship. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?This research project provides training primarily to myself. This project is under the primary mentorship of Dr. Roger Innes at IU and in collaboration with Dr. Thomas Baum at Iowa State University (ISU). Dr. Innes has provided several different opportunities for mentorship on this project including one-on-one meetings where I have discussed various aspects of the research, lab meetings which allows me to share a research presentation once every several months, and subgroup meetings where I can give bi-weekly updates on what I have been working on and accomplished since the last meeting. Not directly related to this research project but other important aspects of pre-doctoral training that are outlined in this fellowship include Dr. Innes' training he has provided to me in the peer review process and my inclusion in outreach events that he is involved in related to plant biology and spreading awareness of plant health. Dr. Baum, his group at ISU, and Dr. Innes participate in monthly Zoom meetings where I provide an update on my research and engage in conversation around troubleshooting and next steps. Dr. Innes and Dr. Baum's combined expertise has trained me to think broadly and from the perspectives of both the plant and the nematode. Thetraining activitiesandprofessional developmentthat have stemmed from their mentorship has been beneficial to both the project and to me as a PhD candidate. Othertraining activitiesthat I have been involved in during this past funding period include training an undergraduate student, who is now a research associate in the Innes lab, and a high school student from a local Bloomington, IN school. Both individuals have been able to learn important laboratory skills such as cloning, plant transient expression assays, immunoblotting, and plant and bacterial upkeep. Jessica Foster, the now research associate, has used her mentorship experience with me to become and independent scientist and a "go to" person in the Innes lab as she is fluent in a variety of techniques applicable to plant-pathogen interactions. Grace Choi, the high school student I mentored, used what she learned about transient expression inN. benthamianaand the RUBY marker to design her own research experiment for a national science fair where she placed 3rd. Thesetraining activitiesthat I was part of allowed me to learn how to teach laboratory skills to people with different levels of understanding and experience. Outside of theprofessional developmentI have achieved from mentoring, it has brought me great enjoyment and excitement to be able to help foster the next generation of scientists. As part of theprofessional developmentduring this past reporting period, I had the opportunity to attend the American Phytopathological Society meeting in Pittsburgh, PA. I presented a poster that included some of the preliminary results of this work, attended various talks, and attended an industry-graduate student lunch. Since submitting this fellowship proposal where I stated I was interested in pursuing a tenured-track faculty position in academia, I have had the pleasure of learning about different career paths in my field. I learned that industry provides me many different aspects I hope to have in a future career after finishing my PhD. At the lunch, I was able to talk with personnel from several different companies, most of which work on very different areas of plant health. This motivated me to learn more about jobs in industry and how the skills I have learned from this project and my training during my PhD could be applied to these different types of positions. At the meeting, I also met another individual through a colleague who works in industry in Raleigh, NC. Over the holidays, she had arranged lunch for her and I to discuss her experiences transitioning from an academic environment to an industry position, her role at the company, and briefly on the types of projects worked on. I then got a tour of both of their campuses and met the team leader of the plant transformation team. This experience has allowed me to think about how I can apply what I have accomplished, learned, and become an expert in through this research project and apply it to a successful rewarding career. How have the results been disseminated to communities of interest?The direct results of this research have been communicated to the Plant Health research community through my poster presentation and networking at the American Phytopathological meeting mentioned several times throughout this report. Additionally, I participated in Indiana University's annual Science Fest, where I had the opportunity to volunteer my time at the Innes lab booth "Plants Get Sick Too." Here I discussed in broad terms what I do as a plant biologist, my overall research goals, and why these kind of topics in agriculture are important to everyone. This increases the awareness of plant pathogens and their overall impact on our food security. It is especially rewarding to discuss the types of avenues scientists and government are pursuing to address these problems. This is especially important and related to this research as there are many misconceptions about genome engineering in plants. As my work focuses on developing a foundation required for these types of pathogen prevention strategies, it has been excellent to share this information with the public. What do you plan to do during the next reporting period to accomplish the goals?During this next reporting period, I will be finishing the immune suppression assays listed under objective one. More specifically, I will be assaying other aspects of PTI such as ROS burst and defense marker gene expression to further confirm these results for publication purposes. As part of my ETI suppression assays, I will quantify the cell death that I have qualitatively collected using an electrolyte leakage assay. With the data that I have already collected and conclusions that I am beginning to make regarding the biology of my SCN effector proteins, I will prepare a manuscript describing my findings. I have already begun to put together an outline for this paper and I anticipate submission of a manuscript during the middle of this funding period. Under my second objective, I will continue performing my miniTurbo experiments in composite soybean roots. Optimization of these methods has already been accomplished and no further complications are expected. I have already begun on this and am now waiting on mass spectrometry data for Hetgly13995 and Hetgly10202. Once I receive this data, I will analyze it as I did with the first dataset of Hetgly22189 and Hetgly14678, clone putative targets from root cDNA that I already have, and determine whether these are true interactions. I am anticipating that by mid-reporting period, I will then have targets confirmed for at least two of my effector proteins. I will then prepare the appropriate CRISPR constructs to knockout these targets in transgenic soybean roots for nematode infection assays. During the next reporting period I am planning to travel to the Baum lab at ISU to learn nematode infection assays and other techniques that the Innes lab lacks expertise in. This will allow me to successfully accomplish my second objective. It will also provide me with the opportunity to work in a different research environment and meet new individuals within my field. I will be presenting at two conferences during this reporting period, which include the IS-MPMI meeting in Providence, RI and the APS meeting in Denver, CO. These conferences will allow me to communicate my research findings, expand my network, and will provide various opportunities for career development. Additionally, I will be attending the USDA PD meeting via Zoom in March as part of the fellowship requirements.
Impacts
What was accomplished under these goals?
Soybean is one of the most economically significant crop plants worldwide as it is a primary source of protein for both animals and humans. However, soybean yield is threatened annually by a variety of pest and pathogens in the environment. The Soybean Cyst Nematode (SCN) is the most damaging pathogen of soybean and results in over $1 billion of crop loss each year. Current methods to prevent SCN infection are limited, and resistant varieties of soybean are losing efficacy due to the nematode's ability to rapidly develop ways to overcome the plant's defense responses. Due to this, new methods to prevent SCN infection are in high demand. To address this problem in agriculture, we must have a deep understanding of the SCN-soybean interaction. It is widely known in the research community that one of the most important aspects of SCN infection includes salivary secretions into the plant cell. In these secretions are proteins known as effectors. Effector proteins are known to hijack and reprogram host cells for the pathogen's benefit. In the case of SCN, these secretions facilitate the suppression of plant immune responses and the remodeling of the plant cell into a permanent feeding site. Many putative effector proteins of SCN, and other plant parasitic nematodes, have been identified but have yet to be characterized. As effector proteins secreted by SCN are known to be one of the most important aspects of infection, I have selected 11 uncharacterized SCN effector proteins with the overall goal of understanding how these proteins function during infection. I have now shown that several of these SCN effector proteins interfere with plant immune responses. These proteins that function in immune suppression have been prioritized for experiments that will allow me to identify the specific plant proteins they target. Initial experiments have identified several putative plant targets with interesting links to SCN infection. I am now testing whether these are true targets of SCN effectors. Identifying the plant protein(s) targeted by the effector protein will be the first step in understanding which plant processes are manipulated by the nematode. More broadly, plant parasitic nematodes are known to infect other economically significant crop plants such as rice, wheat, and sorghum. The combined expertise from Dr. Baum and Dr. Innes will allow me to critically think from both the perspective of the nematode and the plant to illustrate the complex interactions. This research will ultimately provide essential knowledge for developing new varieties of soybean that are resistant to SCN, which ultimately improves soybean health and food security in the USA and world-wide. Objective 1. Determine whether soybean cyst nematode effector proteins function in suppressing plant immune responses. UnderObjective 1, I have generated and confirmed functionality of all constructs and bacterial strains required to carry out the proposed experiments. I have screened 4 of my effectors for suppression of pattern-triggered immunity (PTI) and 5 for the suppression of effector-triggered immunity (ETI). Using a callose assay in soybean leaves, I have shown that 2 of my effectors (Hetgly14677 and Hetgly15757) suppressPseudomonas syringae-induced callose. The callose was stained with aniline blue, imaged with an epifluorescence microscope, and quantified in Image J. A one-way ANOVA test was performed to show significance of the quantified callose area.My data has shown that inactivating the protease domain of Hetgly15757 did not significantly influence the effector's callose-suppression activity. This suggests that the binding of Hetgly15757to a specific plant target is sufficient to suppress callose deposition.I also performed ETI suppression assays using a transient assay inNicotiana benthamianaleaves. In brief, I co-expressed the effector candidate with an auto active resistance gene that upon expression induces programmed cell death. If the effector functions in ETI suppression, cell death does not occur. I have visualized and imaged these leaves under both white and ultraviolet light. I have shown that one of my effector candidates (Hetgly22189) suppresses the RPS5-mediated cell death in three independent trials. I found that inactivation of the effector's protease activity inhibited its ability to suppress cell death.The combined activities and data collected under my first objective have allowed me to identify 3 SCN effectors with immune suppression activity. Thekey outcomesof the activities, data collected, and analysis of results underObjective 1have identified several SCN effector candidates with key roles in the infection process that are specifically involved in immune suppression. These results further illustrate the SCN effector repertoire and emphasizes the importance of suppressing plant immune responses to carry out infection and maintain the nematode's feeding site. Overall, the activities, data collected, and analyses under my first objective have given me an initial list of SCN effector proteins to prioritize forObjective 2. Objective 2. Identify the soybean proteins targeted by soybean cyst nematode effectors and assess how knockout of these targets influence the phenotypes associated with infection. I have generated the required constructs needed to carry out biotin proximity labeling using miniTurbo for 6 of my top priority effector candidates. Top priority candidates were determined based upon theresults ofObjective 1and expression data from Dr. Baum's group. I initially proposed performing miniTurbo in soybean protoplasts. However, initial experiments resulted in low transfection efficiency and protein concentration not suitable for miniTurbo. As an alternative approach, I have generated soybean composite plants usingAgrobacterium rhizogenesstrains that deliver T-DNA encoding the SCN effector with a miniTurbo fusion protein and the RUBY screenable marker. The composite plants and the RUBY marker, which produces visible betalain in transgenic tissue, yield sufficient protein concentrations for biotin-based proximity labeling. I have shown that I am able to express and generate the SCN effectors in the soybean roots. However, the active form of Hetgly15757 was found to be toxic to the plant while the inactivate mutant successfully produced transgenic roots. Additionally, composite soybean roots expressing Hetgly13995, showed a unique phenotype that appeared approximately 3 weeks post inefection. The roots expressing Hetgly13995 were positive for the RUBY marker but appeared to be browning and felt brittle but continued to grow. I have now performed the miniTurbo experiments in composite soybean roots with two of my SCN effectors. Western blot analysis confirmed biotinylation of plant proteins in the composite soybean roots upon exogenous biotin application.Biotinylated proteins were then affinity purified using streptavidin beads. Using mass spectrometry to identify the purified proteins, I found several candidate targets that have interesting links to SCN infection and resistance. Using cDNA that I generated from soybean root RNA, I have cloned several of these putative targets for additional experiments to confirm putative interactions. Thekey outcomesof the activities, data collected, and analysis of results underObjective 2have included (i) the optimization of a suitable method for performing miniTurbo experiments in soybean, the native host of SCN, (ii) the identification oftwo interesting phenotypes (the effect of Hetgl15757 protease activity on transgenic root generation and the brittle black transgenic roots that express Hetgly13395), and (iii) several potential targets identified for two of my SCN candidates (Hetgly14678 and Hetgly22189). Training and career development objectives are described in the next section of this report.
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