Source: UNIVERSITY OF CALIFORNIA, DAVIS submitted to NRP
INVESTIGATING THE ROLE OF A TANDEM KINASE-PSEUDOKINASE IN PLANT DEFENSE
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
COMPLETE
Funding Source
AFRI COMPETITIVE GRANT
Reporting Frequency
Annual
Accession No.
1023831
Grant No.
2020-67013-32577
Cumulative Award Amt.
$406,680.00
Proposal No.
2020-08282
Multistate No.
(N/A)
Project Start Date
Sep 1, 2020
Project End Date
Aug 31, 2024
Grant Year
2020
Program Code
[A1171]- Plant Biotic Interactions
Recipient Organization
UNIVERSITY OF CALIFORNIA, DAVIS
410 MRAK HALL
DAVIS,CA 95616-8671
Performing Department
Plant Pathology
Non Technical Summary
This project supports the mission of the Agricultural Experiment Station by addressing the Hatch Act area(s) of: plant and animal production, protection, and health; molecular biology; biotechnologyStripe rust is a devastating fungal disease that threatens global wheat production, with estimated global losses of around 1 billion dollars per year. Recently, the WTK1 gene was identified from a wild wheat relative that provides broad-spectrum resistance against the fungal pathogen causing stripe rust. Despite the importance of this disease, how WTK1 confers disease resistance remains unknown. This grant will focus on identifying how WTK1 perceives the fungal pathogen causing stripe rust and signals inside wheat cells to confer disease resistance using cutting-edge protein-protein and genome editing technologies. A greater understanding of WTK1-mediated disease resistance will provide important mechanistic insight into signaling networks in plant disease resistance. A key goal of this project is to integrate research with the teaching and training of a diverse student body. The PI will collaborate with a local middle school to develop hands-on plant laboratory modules that will utilize wheat genetics to illustrate the connection between genotype and phenotype. These laboratory modules will be implemented in the 7th grade science curriculum, will be inexpensive, sustainable, and reach >1,000 students during the course of the project.
Animal Health Component
10%
Research Effort Categories
Basic
90%
Applied
10%
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
2124020110250%
2061549104050%
Knowledge Area
212 - Pathogens and Nematodes Affecting Plants; 206 - Basic Plant Biology;

Subject Of Investigation
1549 - Wheat, general/other; 4020 - Fungi;

Field Of Science
1040 - Molecular biology; 1102 - Mycology;
Goals / Objectives
Stripe rust, caused by Puccinia striiformis f. sp. tritici (Pst), is a devastating fungal disease threatening global wheat production, with estimated global yield losses of around one billion dollars per year. The Yr15 resistance gene derived from wild emmer wheat, Triticum dicoccoides, provides broad-spectrum resistance against Pst. We have recently cloned Yr15, which encodes a protein with tandem kinase-pseudokinase domain architecture, and designated it Wheat Tandem Kinase 1 (WTK1). Furthermore, we have discovered that tandem kinase-pseudokinases (TKPs) represent a novel protein family widespread across the plant kingdom. The majority of known plant disease resistance genes encode race-specific, nucleotide-binding leucine-rich-repeat receptors (NLRs), which have been extensively studied over the last 30 years. However, broad-spectrum resistance genes are encoded by various protein families that must function through novel mechanisms to confer resistance. How these noncanonical resistance genes signal to provide disease resistance remains unknown. In this proposal, we seek to understand WTK1 activation and elucidate WTK1 signaling networks. The single TKP found in the Arabidopsis genome evolved by duplication and its function in disease resistance is unknown. Thus, understanding of TPK function in a non-model organism will reveal important mechanistic insights on the diversity of immune signaling. We will work on polyploid wheat in order to investigate WTK1-mediated resistance (as representative member of TKP family) and advance our understanding of novel plant defense mechanisms.The Objectives of this application are:I. Identify and functionally investigate the role of WTK1 and associated proteins. The importance of WTK1 domain architecture and enzymatic activity will be analyzed. WTK1 protein complexes will be purified from wheat protoplasts and the complex members will be identified by mass spectrometry. WTK1 associated proteins will be validated independently and their function will be investigated using genome editing technology, targeting both A and B genomes of tetraploid wheat.II. Analyze WTK1 signaling networks. An RNASeq approach will be used to transcriptionally profile susceptible (Kronos) and resistant (Kronos+WTK1) tetraploid wheat after infection with Pst. We hypothesize that WTK1-mediated recognition will result in significant transcriptional reprogramming. The identification of WTK1 signaling networks will reveal WTK1 pathways facilitating cell death and resistance. RT-qPCR will be used to validate the expression pattern of selected differentially expressed transcripts. The role of selected signaling nodes for WTK1-mediated resistance will be independently validated by genome editing technology.III. Functional validation of the WTK1-interactome. Aims I & II will result in a list of candidate proteins that are associated with WTK1 or important for WTK1-mediated defense. These proteins will be validated independently, and their function will be investigated using genome editing technology, such as CRISPR, targeting both A and B genomes of tetraploid wheat. We will prioritize for validation proteins that appear in both lists from Aim I & II.
Project Methods
The methods include a combination of molecular biology, identification of protein-protein interactions through protein complex purification and mass spectrometry, and genome editing in wheat to investigate the importance of specific loci. Experiments will be repeated at least three times and blinded when possible. Statistical analyses will be used to determine significant differences. Efforts will include classroom instruction, outreach to the general public through K-12 education, and practicum research experiences at the graduate and undergraduate level. Project success will be evaluated by key milestones of success including: completion of protein-protein interaction studies, generation of genome edited lines, and development of laboratory modules for K-12 education. Project success will also be evaluated by publications in peer-reviewed journals.

Progress 09/01/20 to 08/31/24

Outputs
Target Audience:The project supported training for students and scientists at various career stages, resulting in new skills in plant biology, advanced genetic tools, and disease research. We also engaged with local K-12 schools and the community to inspire their interest in becoming a scientist, communicate results and educate the public on topics like genome editing and plant immunity. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?The project supported training for students and scientists at various career stages, resulting in new skills in plant biology, advanced genetic tools, and disease research. We also engaged with local K-12 schools and the community to inspire their interest in becoming a scientist, communicate results and educate the public on topics like genome editing and plant immunity. How have the results been disseminated to communities of interest?The broader public benefited from our project in several ways: Enhanced Food Security: By studying resistance to stripe rust and wheat blast, major threats to wheat production, we are contributing to the development of disease-resistant wheat varieties. Genetic resistance reduces reliance on chemical fungicides and enhances sustainable agricultural practices. Educational Outreach: We engaged with K-12 students and educators through hands-on learning modules and presentations, fostering interest in science, technology, engineering, and mathematics (STEM) fields and raising awareness about the importance of plant health and sustainable agriculture. Workforce Development: The project trained the next generation of scientists and agricultural professional with skills and knowledge to address future challenges in crop protection. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Our project made advancements in how wheat tandem kinases work to protect plants from fungal pathogens. By studying how different tandem kinases function, we are uncovering how plant immunity works with implications for receptor deployment and engineering. 1. Understanding how tandem kinases work in wheat: We demonstrated that WTK1 is an active protein that can modify itself and potentially other proteins to initiate plant defenses. Using advanced methods, we identified wheat plants that successfully produce WTK1, enabling us to study its interactions with other cellular components. RWT4 is another wheat tandem kinase. We demonstrated that RWT4 is an active protein and can directly bind a corresponding effector, serving as an immune sensor. 2. Mapping Defense Signals: We completed a detailed timeline of how wheat plants respond to the stripe rust fungus. By comparing plants with and without WTK1, we identified patterns of gene activation that occur after the plant recognizes the pathogen. This knowledge helps us understand the sequence of events leading to a successful defense. 3. Prevalence of tandem kinases: We generated an atlas of tandem kinases across the plant kingdom. An examination of 104 plant species' genomes uncovered 2,682 tandem kinases. This study demonstrates the widespread occurrence of this family in the plant kingdom. This information can be used to identify new tandem kinases to deploy for disease resistance.

Publications

  • Type: Peer Reviewed Journal Articles Status: Published Year Published: 2024 Citation: Li Y, Wei ZZ, Sela H, Govta L, Klymiuk V, Roychowdhury R, Chawla HS, Ens J, Wiebe K, Bocharova V, Ben-David R, Pawar PB, Zhang Y, Jaiwar S, Moln�r I, Dole~el J, Coaker G, Pozniak CJ, Fahima T. 2024. Dissection of a rapidly evolving wheat resistance gene cluster by long-read genome sequencing accelerated the cloning of Pm69. Plant Communications. 5(1):100646
  • Type: Peer Reviewed Journal Articles Status: Published Year Published: 2023 Citation: Fahima T. and Coaker G. 2023. Pathogen perception and deception in plant immunity by kinase fusion proteins. Nature Genetics, 55(6):908-909
  • Type: Conference Papers and Presentations Status: Submitted Year Published: 2024 Citation: Sung Y-C., Li Y., Baik S., Fahima T., Coaker G. 2024. Investigating the activation model of a wheat tandem kinase upon effector recognition from Magnaporthe oryzae pathotype Triticum. 32nd Fungal Genetics Conference. March 12-17th 2024. Poster presentation. Asilomar, CA, United States.
  • Type: Conference Papers and Presentations Status: Submitted Year Published: 2023 Citation: Govta L., Sela H., Govta N., Potapenko E., Coaker G., Fahima T. Durum wheat immune responses activated by WTK1 (Yr15) against stripe rust. 5th Virtual Durum Meeting, Online conference, 10-11 May 2023. Oral talk.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2023 Citation: Sung Y-C., Li Y., Baik S., Fahima T., Coaker G. 2023. Wheat Tandem kinase with a kinase-pseudokinase protein architecture can recognize its corresponding effector in rice protoplasts and trigger cell death immune response. 2023 IS-MPMI Congress. July 16-20th 2023. Poster presentation. Providence, RI, United States
  • Type: Conference Papers and Presentations Status: Published Year Published: 2023 Citation: Govta L., Govta N., Sela H., Coaker G., Fahima T. Activation of wheat tandem kinase 1 induces expression of genes involved in pathogen recognition, signal transduction, and hypersensitive cell death. 12th International Congress of Plant Pathology, France, Lyon, 20-25 August 2023. Poster presentation.
  • Type: Conference Papers and Presentations Status: Submitted Year Published: 2023 Citation: Reveguk T., Fatiukha A., Potapenko E., Sela H., Klymiuk V., Pozniak C., Wicker T., Coaker G., Fahima T. Diversity of tandem kinase proteins across the plant kingdom. 12th International Congress of Plant Pathology, France, Lyon, 20-25 August 2023. Poster presentation.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2023 Citation: Fahima T., Li Y., Wei Z.Z., Sela H., Govta L., Klymiuk V., Pawar P., Roychowdhury R., Borzov O., Roi Ben-David R., Chawla H.S., Wiebe K., Istv�n I., Dole~el J., Coaker G., Pozniak C. Dissection of a rapidly evolving wheat NLR resistance gene cluster by ont long-read genome sequencing facilitated the cloning Pm69. 12th International Congress of Plant Pathology, France, Lyon, 20-25 August 2023. Poster presentation.


Progress 09/01/22 to 08/31/23

Outputs
Target Audience:The project supported training for students and scientists at various career stages, resulting in new skills in plant biology, advanced genetic tools, and disease research. We also engaged with local K-12 schools and the community to inspire their interest in becoming a scientist, communicate results and educate the public on topics like genome editing and plant immunity. Changes/Problems:This project is jointly funded by NSF and USDA as an NSF-BSF project. Our partner, tzion Fahima is in Israel. due to the war with hamas, research in his lab was delayed beacuse Israeli citizens had to deploy to the military and scientists from other countries left. Therefore, transcriptional profiling was delayed. However, we are still on-track to complete the project in 2024. What opportunities for training and professional development has the project provided?The project supported training for graduate students and a postdoctoral scientist in plant biology, advanced genetic tools, and disease research. Trainees attended scientific meetings and presented their results in oral and poster format. We also engaged with local K-12 schools and the community to inspire their interest in becoming a scientist, communicate results and educate the public on topics like genome editing and plant immunity. How have the results been disseminated to communities of interest?Educational Outreach: We engaged with K-12 students and educators through hands-on learning modules and presentations, fostering interest in science, technology, engineering, and mathematics (STEM) fields and raising awareness about the importance of plant health and sustainable agriculture. What do you plan to do during the next reporting period to accomplish the goals?We will focus on completing transcriptional profiling of wheat after tandem kinase activation. We will also submit two manuscripts focused on tandem kinase activation and the atlast of tandem kinases aacross the plant kingdom. Expirimental efforts will focus on adressing peer review comments.

Impacts
What was accomplished under these goals? Our project made advancements in how wheat tandem kinases work to protect plants from fungal pathogens. By studying how different tandem kinases function, we are uncovering how plant immunity works with implications for receptor deployment and engineering. Understanding how tandem kinases work in wheat: RWT4 is a wheat tandem kinase. We demonstrated that RWT4 is an active protein and can directly bind a corresponding effector, serving as an immune sensor. Prevalence of tandem kinases: We generated an atlas of tandem kinases across the plant kingdom. An examination of 104 plant species' genomes uncovered 2,682 tandem kinases. This study demonstrates the widespread occurrence of this family in the plant kingdom. This information can be used to identify new tandem kinases to deploy for disease resistance.

Publications

  • Type: Conference Papers and Presentations Status: Published Year Published: 2022 Citation: Sung Y-C., Zhu J., Fahima T., Coaker G. 2022. The activation model of the stripe rust resistance gene wheat tandem kinase 1 (WTK1) using in vitro kinase activity assays. Plant Health 2022. August 6-10th 2022. Poster presentation. Pittsburgh, PA, United States.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2022 Citation: Govta, L., Sela, H., Coaker, G., Fahima, T. Transcriptional reprogramming activated by Wheat Tandem Kinase 1 in response to stripe rust infection. International conference From Seed to Pasta IV. Bologna-Italy, 26-29 October 2022. Poster presentation.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2023 Citation: Govta, L., Sela, H., Govta, N., Potapenko, R., Coaker, G., Fahima, T. Plant Immune Responses Activated by Wheat Tandem kinase1 against Stripe Rust Disease of Wheat. International Plant and Animal Genome Conference / PAG 30 (13-18 January 2023, San Diego, USA). Oral talk.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2023 Citation: Govta, L., Sela, H., Govta, N., Potapenko, R., Coaker, G., Fahima, T. Study of transcriptional reprogramming triggered by Wheat Tandem Kinase 1 in response to stripe rust infection. The Israeli Society of Plant Sciences conference, Weizmann Institute of Science, Israel, February 6th, 2023. Poster presentation.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2023 Citation: Govta, L., Sela, H., Govta, N., Potapenko, R., Coaker, G., Fahima, T. Differentially expressed wheat genes activated by Wheat Tandem Kinase 1 in response to stripe rust infection. 10th ILANIT/ FISEB Conference, Eilat, Israel, 20-23 February 2023. Flash talk presentation.


Progress 09/01/21 to 08/31/22

Outputs
Target Audience:The target audience includes other scientists, the general public, and junior high students. A key goal of this project is to integrate the completion of the proposed research with the teaching and training of a diverse student body. Our partnership with Tzion Fahima has facilitated international US-Israel collaborations. Over the last year, we have supported a postdoctoral scientist and an undergraduate student to work on the project. The PI is also involved in co-mentoring one PhD student at the University of Haifa (paid by the collaborating laboratory). Outreach activities to junior high school students in California were performed focused on plant genotyping, genetics, and disease. Outreach activities for K-12 students also have been organized, including outreach to Explorit's Girls in STEM and presentations at high schools in the California Bay area to discuss plant pathology research. In addition to the activities described above, the PI also attended question and answer sessions about her research during a "meet the scientist" virtual field trip for junior high students in California Scientific results were shared with other researchers in publications as well as scientific talks. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?This project has provided opportunities for training and professional development for one PhD student and postdoctoral scientist. The PhD student is based at the University of Haifa, Israel in Tzion Fahima's laboratory, who is the collaborating scientist on this grant. The student has obtained training through one on one meetings with both Tzion Fahima and Gitta Coaker through Zoom. The student has had the opportunity to discuss her research project multiple times in both groups and present data. This project has also provided an opportunity for training and professional development for one postdoctoral scientist, in Gitta Coaker's laboratory. The postdoc has also had the opportunity to present his results during meetings with both Tzion Fahima and Gitta Coaker. The postdoc has also developed an individual development plan in conjunction with Gitta Coaker. Both the student and postdoc have attended international meetings and presented their results over the last year. How have the results been disseminated to communities of interest?Scientific results were shared in journal articles, conferences and invited presentations as detailed in the "products" section. What do you plan to do during the next reporting period to accomplish the goals?During the next reporting period, research efforts will be focused performing proximity labeling of WTK1-BirA transgenic lines and verifying their resistance to Puccinia striiformis f. sp. tritici. Research efforts will also focus on characterizing the importance of individual domains of TKPs for activity and effector recognition. Current WTK1 interacting proteins identified by yeast-two hybrid as well as interesting signaling proteins will be validated and investigated using genome editing. The completion of these experiments will result in a more mechanistic understanding of WTK1 targets and activation at the receptor level. Gitta Coaker will host a research scientist from Tzion Fahima's laboratory for a six-month period to assist in plant genotyping and phenotyping efforts.

Impacts
What was accomplished under these goals? Objective 1. Identify and functionally investigate the role of WTK1 and associated proteins. Functionally investigate the role of WTK1 WTK1's kinase-pseudokinase domain architecture represents an unexplored family of proteins across the plant kingdom. Plant pseudokinases are emerging as important players in diverse biological processes and represent ~10% of the kinase domains in higher eukaryotes. In order to investigate the WTK1 protein, we have generated high confidence structural models of WTK1 to identify regions of interest based on EMS mutations and other in silico analyses. WTK1 and each domain have been expressed in different expression platforms, with some promising results in higher order eukaryotic expression systems. A monocot protoplast assay has been established enabling expression of WTK1 and other tandem kinase proteins for functional analyses. Identify WTK1 associated proteins In order to identify WTK1 associated proteins, a proximity-labeling approach using the TurboID system will be used. Recent advancements using the GRF-GIF system have enabled more reliable and rapid wheat transformation (Nature Biotechnology 38:1274-1279). We are using the next generation biotin ligase TurboID, which will provide enhanced labeling and specificity in plants and has been demonstrated to work in monocots (Frontiers in Plant Science, 8:1-10; eLife,8:e4786). We have generated transgenic wheat expressing WTK1 fused to a biotin ligase, as well as controls and have identified transgenic lines that express the WTK1 fusion. The lines are currently being advanced in the greenhouse to test for their function in disease assays and advance material for proximity labeling. Objective 2. Analyze WTK1 signaling networks. To investigate WTK1 immune signaling, we have conducted transcriptional profiling in wheat upon infection with the fungal pathogen Puccinia striiformis f. sp. tritici (Pst). Transcriptome time-course analysis occurred over 7 days using the resistant genotype Kronos+WTK1 compared with its susceptible Kronos near-isogenic line (NIL) after inoculation with Pst. Eighty samples were processed and analyzed for time-course gene expression data analysis. Clusters predominantly upregulated in Kronos+WTK1 and were enriched in genes involved in pathogen recognition, signal transduction, phytohormone production, and hypersensitive cell death response. These data indicate that WTK1 perception results in plant immune activation. Objective III. Functional validation of the WTK1 interactome In collaboration with another research group, two interacting proteins for WTK1 were identified by a yeast two-hybrid screen and we are currently testing their role in disease resistance using genome editing. We are also performing genome editing of interesting signaling genes identified in Objective 2.

Publications

  • Type: Conference Papers and Presentations Status: Published Year Published: 2021 Citation: Govta, L., Klymiuk V., Coaker G., Fahima T. 2021. The wheat yellow rust resistance gene Yr15 is a member of the TKP protein family. COST-INDEPTH final meeting, August 18-21th 2021. From chromatin domains to nuclear compartments in model plants and crop species. Poster presentation.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2022 Citation: Sung YC, Li Y, Fahima T, Coaker G. The activation model of the stripe rust resistance gene wheat tandem kinase 1 (WTK1) using in vitro kinase activity assays. Poster Presentation. American Phytopathological Society Plant Health Meeting, Pittsburg, PA, August 6-10, 2022, P-265.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2021 Citation: Coaker, G. 2021. The 6th International Conference on Biotic Plant Interactions (6th ICBPI). Shanghai, China October 10th. Keynote Speaker
  • Type: Conference Papers and Presentations Status: Published Year Published: 2022 Citation: Fahima, T. 2022. Protection of Food Crops from Devastating Diseases: A Basic for Increased Food Security and Environmental Safety Week of Livelihoods and Agriculture, The Israel Pavilion, Expo Dubai 2020, Dubai, February 20th, 2022. Invited Speaker.
  • Type: Other Status: Published Year Published: 2021 Citation: Coaker, G. 2021. Investigating the role of plant immune perception and pathogen virulence. China Agricultural University, Seminar. October 12
  • Type: Other Status: Published Year Published: 2021 Citation: Coaker, G. 2021. 2021 Eva J. Pell Interdisciplinary Lecture on Plant Health at Penn State, Distinguished Speaker. Penn State University. Dec 6
  • Type: Other Status: Published Year Published: 2022 Citation: Coaker, G. 2022. Stanford University. Plant Biology Department seminar. April 29, 2022


Progress 09/01/20 to 08/31/21

Outputs
Target Audience:The target audience includes other scientists, the general public, and junior high students. A key goal of this project is to integrate the completion of the proposed research with the teaching and training of a diverse student body. Our partnership with Tzion Fahima has facilitated international US-Israel collaborations. Over the last year, we have supported a technician and have recently hired a postdoctoral scientist to work on the project. The PI is also involved in co-mentoring one PhD student at the University of Haifa (paid by the collaborating laboratory). Outreach activities to junior high school students in California, Arizona, and New York were performed. The PI led a month-long virtual plant laboratory focused on plant genotyping, genetics, and disease. Students determined the genotype of the seeds based on their phenotypic properties. Students also researched and identified any diseases observed during seed germination or later growth. In addition to the activities described above, the PI also attended question and answer sessions about her research during a "meet the scientist" virtual field trip for junior high students in California. Scientific results were shared with other researchers in publications as well as scientific talks. Changes/Problems:The project started shortly after the country shut down due to the COVID-19 pandemic. Therefore, during the first six months of the project, scientists were either unable or severely impacted in their ability to perform research in person. At UC Davis, we are only in the last two months at full research capacity on campus. In addition, we were restricted in hiring new personnel at the start of the project as they would not be able to work in person. Therefore, we initially focused on in silico analyses of WTK1, which was fruitful and identified regions within the resistance protein for targeted laboratory investigation. We have also decided to use a proximity labeling approach as opposed to standard immunoprecipitation mass spectrometry to identify WTK1 interacting partners in planta. We decided to use this approach due to recent advancements in next generation proximity labeling (TurboID) in plants, enabling greater efficiency and specificity of biotin labeling, especially for transient protein-protein interactions that would be expected for kinase-substrate interactions. Due to the inability to perform research in person for several months during the past year, we have had delays in generating transgenic lines for proximity labeling. Due to the pandemic, the UC Davis transformation facility was also not accepting new orders for a period of time. However, we now have all constructs in hand for transformation and using the GRF-GIF system and anticipate that transgenic lines can be generated and tested within the next year. Due to uncertainty surrounding the ability to perform research in person, hiring new researchers for the project was delayed. Over the past 2-3 months, a more positive and clear future for full return to research has occurred in the UC system. Therefore, we have interviewed and hired an excellent postdoctoral scientist who started to work full time on the project. Another scientist will be shared between the Fahima and Coaker lab (50/50) for the project and come to UC Davis in 2022. Due to hiring delays, we did not spend as much as anticipated on personnel, which will allow for more individuals to simultaneously work on the project in future years. What opportunities for training and professional development has the project provided?This project has provided opportunities for training and professional development for one PhD student and one technician. The PhD student is based at the University of Haifa, Israel in Tzion Fahima's laboratory, who is the collaborating scientist on this grant. The student has obtained training through one on one meetings with both Tzion Fahima and Gitta Coaker through Zoom. The student has had the opportunity to discuss her research project multiple times in both groups and present data. This project has also provided an opportunity for training and professional development for one technician, in Gitta Coaker's laboratory. The technician has also had the opportunity to present her results during meetings with both Tzion Fahima and Gitta Coaker. The technician also developed an individual development plan in conjunction with Gitta Coaker. How have the results been disseminated to communities of interest?Outreach activities to junior high school students in California, Arizona, and New York were performed. The PI led a month-long virtual plant laboratory focused on plant genotyping, genetics, and disease. Students determined the genotype of the seeds based on their phenotypic properties. Students also researched and identified any diseases observed during seed germination or later growth. In addition to the activities described above, the PI also attended question and answer sessions about her research during a "meet the scientist" virtual field trip for junior high students in California. Scientific results were shared in journal articles, conferences and invited presentations as detailed in the "products" section. What do you plan to do during the next reporting period to accomplish the goals?During the next reporting period, research efforts will be focused on advancing WTK1-BirA transgenic lines and verifying their resistance to Puccinia striiformis f. sp. tritici. Transgenic lines that exhibit resistance will be used to identify phosphorylated targets using proximity labeling and mass spectrometry. Research efforts will also focus on characterizing WTK1 in vitro the role of domain architecture and both insertion regions with respect to kinase activity. The completion of these experiments will result in a more mechanistic understanding of WTK1 targets and activation at the receptor level. During the next reporting period we will also focus on finishing time course RNASeq analyses to transcriptionally profile susceptible (-WTK1) and resistant (+WTK1) wheat after infection with Pst. The resulting data will be used to analyze WTK1 signaling networks and will help guide a list of targets for functional validation.

Impacts
What was accomplished under these goals? Objective 1. Identify and functionally investigate the role of WTK1 and associated proteins. Functionally investigate the role of WTK1 WTK1's kinase-pseudokinase domain architecture represents an unexplored family of proteins across the plant kingdom. Plant pseudokinases are emerging as important players in diverse biological processes and represent ~10% of the kinase domains in higher eukaryotes. In order to investigate the WTK1 protein, we have generated high confidence structural models of WTK1 and used this to identify regions of interest based on EMS mutations and other in silico analyses. To investigate the WTK1 protein, we have generated high confidence structural models of WTK1 using the pseudokinase-kinase domains from the protein kinase TYK2 as well as the BIK1 and PBL2 structures. We have mapped EMS mutations blocking resistance onto the WTK1 structural model to identify mutations that may be involved in enzymatic activity as well as potential effector binding and localize to the surface of the protein. WTK1 kinase I domain is a wall-associated kinase, while its kinase II domain is predicted to be inactive and is a member of receptor like cytoplasmic kinases. We aligned both WTK1 domains to known wall-associated kinases and receptor-like cytoplasmic kinases, revealing three extra insertions that are missing in other known kinases, map to the predicted surface of WTK1, and co-localize with three EMS mutations blocking WTK1-mediated resistance. We also investigated additional tandem-kinase proteins involved in disease resistance (WTK3 and RPG1) in silico. Our analyses also revealed that both WTK3 and RPG1 also possess two short regions that lack homology with single domain kinases. In the case of WTK3, these one of these regions borders the deletion required for disease resistance. Taken together, these data indicate that WTK1's short insertions are critical and may be involved in effector or substrate binding. These hypotheses will be tested with purified WTK1 and can serve as a model for function of other tandem kinase proteins. Identify WTK1 associated proteins In order to identify WTK1 associated proteins, a proximity-labeling approach using the TurboID system will be used. Recent advancements using the GRF-GIF system have enabled more reliable and rapid wheat transformation (Nature Biotechnology 38:1274-1279). We are using the next generation biotin ligase TurboID, which will provide enhanced labeling and specificity in plants and has been demonstrated to work in monocots (Frontiers in Plant Science, 8:1-10; eLife, 8:e4786). We have generated constructs for proximity labeling using a single T-DNA to co-express GRF-GIF as well as WTK1 to enable reliable transformation efficiency. We have generated constructs for transformation containing WTK1-V5-BirA, BirA-V5-WTK1, and BIRA-V5 driven by the ubiquitin promoter. These expression of the WTK1 derivatives and the BIRA-V5 negative control were verified using transient expression in N. benthamiana and western blotting. Each construct has been sent for transformation of the susceptible wheat cultivar Kronos in the next two weeks. When transgenic lines are obtained, after feeding with biotin, the WTK1 fusion protein will biotinylate proteins in close proximity, allowing for the capture of in vivo protein-protein interactions and their identification by mass spectrometry. Objective 2. Analyze WTK1 signaling networks. Plant-pathogen interactions have resulted in the co-evolution of defense and counter-defense mechanisms, which involve specific resistance proteins, effectors, and co-factors in the host plants and their pathogens. To investigate the role of WTK1 in the immune signaling network that activates the resistance mechanism conferred by WTK1 we will test TKPs activation that mediates resistance in a non-model organism (wheat) by profiling transcriptional changes that occur upon the infection process of yellow rust caused by the fungus Pst. For this experiment, we have selected homozygous transgenic Kronos+Wtk1 plants that showed no segregation for resistance response at T4 generation. To optimize the experimental conditions for transcriptome analysis we have tested the response of these lines to infection with five different Pst spore concentrations and four different isolates over time, analyzing fungal accumulation by microscopy as well as visual phenotypes. These analyses demonstrated that key interactions occur during the first seven days post inoculation. We have sampled leaf segments from the Kronos and Kronos+Wtk1 and have prepared them for fluorescence microscopy and transcriptome analysis. We send the collected samples for Whole Transcriptome analysis (RNAseq) to identify genes and pathways that were altered after inoculation with Pst isolate #5006 at each time point.

Publications

  • Type: Journal Articles Status: Published Year Published: 2020 Citation: Zhang J, Coaker G, Zhou JM, Dong X. Plant Immune Mechanisms: From Reductionistic to Holistic Points of View. Mol Plant. 2020 Oct 5;13(10):1358-1378.
  • Type: Journal Articles Status: Published Year Published: 2020 Citation: Klymiuk V, Fatiukha A, Raats D, Bocharova V, Huang L, Feng L, Jaiwar S, Pozniak C, Coaker G, Dubcovsky J, Fahima T. Three previously characterized resistances to yellow rust are encoded by a single locus Wtk1. J Exp Bot. 71(9):2561-2572.
  • Type: Journal Articles Status: Published Year Published: 2021 Citation: Klymiuk V, Coaker G, Fahima T, Pozniak C. Tandem protein kinases emerge as new regulators of plant immunity. Mol Plant Microbe Interact. 2021 doi: 10.1094/MPMI-03-21-0073-CR.�
  • Type: Conference Papers and Presentations Status: Published Year Published: 2021 Citation: Coaker, G. 2021. Mechanisms regulating Arabidopsis immune signaling and their translation to crop species. Seminar, SFP924: Molecular Mechanisms regulating yield and yield stability in plants. TUM School of Life Sciences. Freising, Germany. July 8th
  • Type: Conference Papers and Presentations Status: Published Year Published: 2020 Citation: Fahima T., Fatiukha A., Klymiuk V., Pozniak C., Coaker G. 2020. The Discovery of the Tandem Kinase-Pseudokinase (TKP) Protein Family and a Proposed Decoy Model for Their Involvement in Plant Immunity. The Borlaug Global Rust Initiative Online Technical Workshop, October 7-9, 2020.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2020 Citation: Fahima T., Fatiukha A., Klymiuk V., Pozniak C., Coaker G. 2020. Polyphyletic origin of the tandem kinase-pseudokinase (TKP) protein family and a proposed decoy model for their involvement in plant immunity. Oral presentation at the 2nd Meeting of the Israeli Society for Evolutionary Biology, December 9-10, 2020.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2020 Citation: Govta L. Klymiuk V., Fatiukha A., Coaker G., Fahima T 2020. The wheat stripe rust resistance gene Yr15 as a member of tandem kinase-pseudokinase protein family. Poster presentation at the 2nd Meeting of the Israeli Society for Evolutionary Biology, December 9-10, 2020.