Progress 06/01/23 to 05/31/24
Outputs
Target Audience:Our project targeted undergraduate and graduate students, as well as scientists at Universities, Federal Laboratories, and Industry stakeholders. Teachers at secondary schools, community colleges and Universities were also engaged through our iTAG barley education and outreach programs. Dissemination of project results occurred through teacher training, publication in refereed journals and presentation at National, International, and society meetings. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?For Aims 1 and 2 , we recruited 2 new undergraduates to train in bioinformatics and genetics of host - pathogen interactions. For Aim 3, we recruited 6 undergraduate faculty from a variety of Carnegie categories (community colleges, small private schools, and US Air Force Academy). They participated in a 5-day workshop learning how to connect the plant phenotypes to genotype using PCR, restriction digestion, and gel electrophoresis. We recruited another graduate student to complete the GMO exercise and the Central Dogma project. How have the results been disseminated to communities of interest?This project has produced research articles in peer-reviewed journals, genome-level datasets, and updated novel software. The PI, Co-PIs, and students have disseminated research findings at national and international scientific meetings. Undergraduate and high school students have been mentored for future careers in STEM fields. What do you plan to do during the next reporting period to accomplish the goals?For Aim 1, we are revising manuscripts on "Gene Regulatory Networks (GRN) of transcription factor (TF) - mediated control of barley in response to barley powdery mildew, as well as software updates. We are also testing a novel epistasis hypothesis on regulatory control of nucleotide-binding leucine-rich receptors (NLRs). For Aim 2, we are testing high-confidence transcription factors and their cis-regulatory elements via ChIP-Seq, ATAC-Seq and gene silencing assays. For Aim 3, we are addressing a recently uncovered series of misconceptions and skill deficiencies in molecular biology education. During the course of iTAG development and publication we recognized a significant gap in student understanding of the flow of genetic information at the introductory level. In order to address this, we are developing a series of videos that visually describe the central dogma of molecular biology. We will start with DNA replication, then transcription, and finally translation, thus following the flow of information from DNA to protein. These videos and associated manuscripts, produced following universal design principles, will be published and made readily available for instructors at the community college and introductory university levels.
Impacts
What was accomplished under these goals?
AIM 1: Cellular pathways that control plant immunity are guided by complex transcriptional networks. Activation of these networks is often mediated by nucleotide-binding leucine-rich repeat (NLR) immune receptors. To continue to support Aim 1, we used the interaction between an archetype NLR receptor, barley MLA, and the biotrophic powdery mildew fungus, Blumeria hordei (Bh), to interrogate the temporal regulation of immune signaling via computational prediction of a Gene Regulatory Network (GRN). The three algorithms, dynGENIE3, Inferelator, and GRACE, were used to predict a gene regulatory network which comprises 70% of 34603 high-confidence genes in the barley genome, including 1214 transcription factors (TF), 20125 targets, and 54698 interactions. In silico gene knockouts highlighted the most essential TF in the network; these were then selected for antibody design and time-course chromatin immunoprecipitation sequencing, where loss-of-function mutation of the MLA6 NLR was associated with genome-wide disruption of TF-promoter interactions and TF-target gene expression. A newly identified NAM-ATAF1,2-CUC2 (NAC) TF influences the activation of Mla, along with feedback loops linking homeobox (HB), WRKY2 and MADS-box proteins that trigger a dynamic TF network that controls hundreds of genes during fungal infection, including penetration of host epidermal cells and development of haustorial feeding structures. These results suggest a unique auto-regulatory mechanism of the immune receptor that drives the response to powdery mildew over the course of disease progression. AIM 2: Several branches of the plant immune system are known to depend on endomembrane traffic, which pathogens suppress using secreted effectors to promote disease. For functional analysis under Aim 2, in collaboration with Iowa State University and the University of Copenhagen in Denmark, we combined bioinformatic analysis with high-resolution cellular biology to untangle critical interactions among host and pathogens. Proteins need to be shipped, or "trafficked" to specific locations within the cell during the plant life cycle. For example, vacuolar transporters are part of a complex network that enables a plant to react to changing environmental conditions, such as pathogen attack. Research focused on characterization of protein-protien interactions (PPI) that localized to the endoplasmic reticulum (ER), and a set of five critical powdery mildew effectors that differentially impact trafficking of proteins from the ER to the vacuole during powdery mildew infection. We found that the barley powdery mildew fungus uses these effectors that target endomembrane traffic to suppress effector-triggered immunity activated by cytosolic nucleotide-binding leucine-rich receptors (NLRs) via interference with the late endosome pathway. This phenomena is just not restricted to plants, and can be traced back 1.3 billion years to yeast. This key knowledge step can be exploited for next-generation marker assisted selection (MAS) to breed for resistance to new and emerging pathogens. AIM 3: For Aim 3, we hosted the 2023 summer iTAG workshop for community college and university instructors. "Inheritance of Traits and Genes" (iTAG) uses the diverse Oregon Wolfe Barleys in lab and classroom activities to connect visible traits (phenotype) to DNA sequence (genotype). Basic molecular biology techniques such as DNA extraction, polymerase chain reaction, and gel electrophoresis were used to document different types of DNA polymorphisms among plants with different phenotypes. Instructors can then return to their institutions and lead discussions with their students of how scientists associate genotype and phenotype in real life scenarios. Thus, young researchers gain valuable experience in genetic history, cellular pathways and developmental mutations; concepts critical to producing disease resistant crops and livestock. iTAG barley has been used by nearly 50 instructors across the USA in >200 high school and community college biology classes from 2010 to 2024, impacting nearly 5000 students, of which one third were from underrepresented groups from urban to rural communities. During the workshop, feedback was solicited from instructors that teach in higher education as to how the iTAG manual could best serve their needs, as its original version was aimed toward high school classroom instruction. Following the workshop, the course overview and lab manual were published in The Plant Health Instructor at https://www.apsnet.org/edcenter/learningPP/Pages/PHI-E-2023-09-0009.aspx on the American Phytopathological Society (APS) Education Resources website.
Publications
- Type:
Peer Reviewed Journal Articles
Status:
Published
Year Published:
2024
Citation:
Wise, RP, G Fuerst, N Peters, N Boury, L McGhee, M Greene, S Michaelson, J Gonzalez, N Hayes, R Schuck, L Maffin, G Hall, T Hubbard, E Whigham. 2024. iTAG: Interactive Laboratory Exercises to Explore Genotype and Phenotype Using Oregon Wolfe Barley?. The Plant Health Instructor: 24.
- Type:
Peer Reviewed Journal Articles
Status:
Published
Year Published:
2024
Citation:
Li, Z, V Vel�squez-Zapata, JM Elmore, X Li, W Xie, S Deb, X Tian, S Banerjee, HJL J�rgensen, C Pedersen, RP Wise, H Thordal-Christensen. 2024. Powdery mildew effectors AVRA1 and BEC1016 target the ER J-domain protein HvERdj3B required for immunity in barley. Mol Plant Pathol. 2024;25:e13463.
- Type:
Peer Reviewed Journal Articles
Status:
Published
Year Published:
2024
Citation:
Boury, N, M Van den Bogaard, EB Wlezien, N Peters, R Wise. 2024. The Great Petunia Carnage of 2017: A Clicker Case Study Using Petunias to Describe the Effect of Genetic Modification on the Biochemistry of Flower Color and Phenotype in Plants. CourseSource: Evidence-based Teaching Resources for Undergraduate Biology and Physics.
- Type:
Peer Reviewed Journal Articles
Status:
Published
Year Published:
2024
Citation:
Vel�squez-Zapata, V, S Smith, P Surana, AVE Chapman, N Jaiswal, M Helm, RP Wise. 2024. Diverse epistatic effects in barley-powdery mildew interactions localize to host chromosome hotspots. iSCIENCE 27(10), 111013.
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Progress 06/01/22 to 05/31/23
Outputs
Target Audience:Our project targeted undergraduate and graduate students and scientists at Universities, Federal Laboratories, and Industry, in addition to teachers at secondary schools, community colleges and Universities. Dissemination of project results occurred through teacher training, publication in refereed journals and presentation at National, International, and society meetings. Changes/Problems:Due to COVID restrictions early in the project, we will likely be requesting a second no-cost extension in 2024 to finish ChIP-Seq and ATAC-Seq protocols and manuscripts. What opportunities for training and professional development has the project provided?Aim 1: In 2022, Dr. Velásquez-Zapata earned a prestigious DeLill Nasser Professsional Development Award from the Genetics Society of America.Also, for Aim 1, she adapted several state-of-the-art computational approaches to integrate a large-scale RNA-Seq infection time-course of resistant wild type and susceptible immune mutants, expression quantitative trait loci (eQTL) analysis, and the results of her Y2H interactome studies. This earned a cover article in GENETICS in June 2022https://doi.org/10.1093/genetics/iyac056and was the topic of an invitation to present her research at the International Plant & Animal Genome conference in San Diego, CA (January 2023). She also has secured a permanent bioinformatics scientist position at GreenLight Biosciences in Research Triangle Park, North Carolina. Aim 3: Our graduate student EB Wlezien finishedher Ph.D. in Spring 2023 and secured a position at FMC corporation, an agricultural sciences company in Delaware. How have the results been disseminated to communities of interest?This project has produced research articles in peer-reviewed journals, genome-level datasets, and updated novel software. The PI, Co-PIs, and students have disseminated research findings at national and international scientific meetings. Undergraduate and high school students will be mentored for future careers in STEM fields. What do you plan to do during the next reporting period to accomplish the goals?ForAim 1, we are developing manuscripts on "Transcriptome-based gene interaction models reveal epistatic and co-expression modules in the barley-powdery mildew pathosystem" and"Gene Regulatory Networks (GRN) of transcription factor (TF) - mediated control of barley in response to barley powdery mildew. ForAim 2, we are testing high-confidence transcription factors and their cis-regulatory elements via ChIP-Seq, ATAC-Seq and gene silencing assays. For Aim 3, we aredeveloping manuscripts on iTAG training to connect the OWB plant phenotypes to genotype using PCR, restriction digestion, and gel electrophoresis. Also preparingand thecase study, "The great Petunia Carnage of 2017" whichincludes class discussion of regulations governing genetically modified organisms and public opinion of both GMO's and the regulatory process.
Impacts
What was accomplished under these goals?
Further insights into disease resistance signaling were obtained in FY2023. To support Aim 1, the teamcontinued research to validate a genome-wide regulatory networkof 1214 transcription factors, 20125 cellular protein targets, and 54698 interactionstoexplore disease response in large-genome, cereal grains.At the center isthe Mildew locus a (MLA)nucleotide-binding leucine-rich-repeat (NLR)immune receptor, an ancestral protein required for protection against destructive cereal diseases, including powdery mildew, Ug99 stem rust, stripe rust, and rice blast. We are currently using ChIP-Seq and ATAC-Seq to physically validate cis-regulatory element (CRE) time-course predictions for WRKY2, NAC, MADS, and Homeobox (HB), major transcription factors in the gene regulatory network for response to powdery mildew.These results will be leveraged to develop and extend resistance to new and emerging pathogens. In addition, time-course gene expression of barley and thepowdery mildew pathogenwasusedto infer diverse gene effects governed by the MLA immune receptor and two other host genes critical to disease defense,Blufensin1(Bln1) andRequired for Mla6 resistance3 (Rar3).Gene effect models revealed symmetric, suppression and masked epistatis influenced byMla6andBln1 (a situationwhere the expression of one gene is modified by the expression of other genes);anddominanteffects ofMla6andSgt1on thepowdery mildew-influencedbarley transcriptome.From a total of 468 barley NLRs, 366 were expressed and 115 of those were classified under different gene effect models, whichclustered at several chromosome hotspots.Most plant resistance genes deployed in agriculture encodeNLRs. However, the mechanisms by which NLR receptors impart critical functions to plant cells are often targets of pathogen effectors, thus,these discoveries provide a foundation for further research into the complex molecular interactions that control disease resistance in crops. For functional analysis underAim 2,in collaboration with Iowa State University and the University ofCopenhagen in Denmark, weemployed advanced genomic technology known as yeast two-hybrid next-generation interaction screens to discoverthat a pair of powdery mildeweffectors, designatedAVRA1and BEC1016,target the barleyJ-domain protein,HvERdj3B, in the endoplasmic reticulum (ER).HvERdj3B is an ER quality control protein and suppressing its function increased powdery mildew disease. Together, these results suggest that barley innate immunity, that is, preventing powdery mildew penetration into epidermal cells, is dependent on ER quality control, which in turn requires the J-domain proteinHvERdj3B and is regulated by the two effectors.These resultsto identify new components of host disease defense and the pathogen effectors that suppress themwill enable breeders and growers to more effectively use disease resistance to produce better crops. For Aim 3;iPath/iTAGoutreach Goals/Deliverables at the end of the project: Multi-level case studies for biology literacy (based on discoveries from Aims 1 & 2) Undergraduate course modules (based on discoveries from Aims 1 & 2) iTAG/iPathRET whiteboard videos describing concepts in genetics and plant disease Nation-wide dissemination to underrepresented groups What was accomplished under these goals? Multi-level case studies & learning exercises In the summer of 2022 we worked with ISU's research experiences for teachers (RET) program to identify and host a high school teaching fellow to work with Drs Wise, Peters, and Boury to develop PCR tests for identified alleles that can be used to further explore the connections between genotype and phenotype. We have created and class-tested a case study emphasizing the biochemical pathways that connect a given genotype with its phenotype. This case study, "The great Petunia Carnage of 2017" also includes class discussion of regulations governing genetically modified organisms and public opinion of both GMO's and the regulatory process. Thesocioscientificissues (SSI) provide context for the course content of central dogma (DNA is copied to RNA which is the template to make a protein), Mendelian genetics, and basic biochemical principles.This case study was developed in Spring 2022 and field-tested in Spring 2022 (Year 2) and 2023 (Year 3) Undergraduate course modules: AGMO Flipped Classroom activity was submitted to CourseSource and is currently in final revision. Creation ofiTAG/iPathWhiteboard videos:In progress -This video is the first of a series that will include DNA replication, transcription, translation, and a final video connecting genotype (alleles present) to various phenotypes (e.g. disease resistance and susceptibility, 2-row and6 row, etc.) in barley. National Dissemination We have published a learning activity (Potato Late Blight and Southern corn leaf blight jigsaw). The Petunia's Case Study and GMO flipped classroom exercise have been tested and the manuscripts are in preparation. The whiteboard videos, when published, will be available as an Open Education Resource (OER) and be shared with colleagues at 1890 Land Grant and Hispanic Serving schools prior to their official release. (1 video produced in year 2 and 2 produced in year 3).
Publications
- Type:
Book Chapters
Status:
Published
Year Published:
2023
Citation:
Elmore, JM, V Vel�squez-Zapata, RP Wise. 2023. Chapter 19, Next-Generation Yeast Two-Hybrid Screening to Discover ProteinProtein Interactions. In Protein-Protein Interactions: Methods and Protocols, Methods in Molecular Biology, vol. 2690, https://doi.org/10.1007/978-1-0716-3327-4, Shahid Mukhtar (ed.), Springer
- Type:
Book Chapters
Status:
Published
Year Published:
2023
Citation:
Vel�squez-Zapata, V, JM Elmore,RP Wise. 2023. Chapter 20, Bioinformatic Analysis of Yeast Two-Hybrid Next-Generation Interaction Screen Data. In Protein-Protein Interactions: Methods and Protocols, Methods in Molecular Biology, vol. 2690, https://doi.org/10.1007/978-1-0716-3327-4, Shahid Mukhtar (ed.), Springer
|
Progress 06/01/21 to 05/31/22
Outputs
Target Audience:Our project targeted undergraduate and graduate students and scientists at Universities, Federal Laboratories, and Industry, in addition to teachers at secondary schools. Dissemination of project results occurred through secondary school teacher training, publication in refereed journals and presentation at National, International, and society meetings. Changes/Problems: Due to COVID related delays, we will likely be requesting a no-cost extension next spring What opportunities for training and professional development has the project provided?Aim 1: Valeria Velásquez-Zapata defended her PhD in Bioinformatics & Computational Biology in July 2021. She was focused on the informatics and gene regulation part of the project (Specific Aims 1 and 2). She was then hired as a postdoc and has continued in that role. She has now published her second (first author) paper, which earned the June 2022 cover of GENETICS, and has presented at Plant and Animal Genome XXIX, and the GSA Population, Evolutionary, and Quantitative Genetics Conference, the IS-MPMI (virtual) conference, and the International Powdery Mildew Genome workshop (virtual). She has also co-authored several additional manuscripts. To further her professional development, she also earned an Iowa State University Graduate College Research Excellence Award (Presented to top graduates in each field) and a Genetics Society of America DeLill Nasser Professional Development Award. Aim 3: Our other graduate student, EB Wlezien, advanced her professional development outcomes with both procedural knowledge (e.g. how to use graphics programs such as BioRender, how to use education best practices to design lessons and case studies, etc.) and conceptual knowledge on genotype and phenotypic interactions in the Oregon Wolfe Barley (OWB) Informative and Spectacular Subset of Barley populations. She also published a first author paper in CourseSource (referred to above): Wlezien et al. 2022. Role of crop genetic diversity on pathogen impact: The tale of two pathogens. CourseSource. https://doi.org/10.24918/cs.2022.14 How have the results been disseminated to communities of interest?This project has produced research articles in peer-reviewed journals, genome-level datasets, and updated novel software. The PI, Co-PIs, and students have disseminated research findings at national and international scientific meetings. Undergraduate and high school students will be mentored for future careers in STEM fields. What do you plan to do during the next reporting period to accomplish the goals?For Aim 1, we plan to develop a manuscript on the Gene Regulatory Network (GRN) of transcription factor (TF) - mediated control of barley and barley powdery mildew For Aim 2, we plan to test high-confidence transcription factors and their cis-regulatory elements via ChIP-Seq, ATAC-Seq and gene silencing assays. For Aim 3, we expect to 1) Finish central dogma videos 2) Classroom test these videos 3) Update iTAG adding learning objectives and assessments 4) Host iTAG workshop for community college instructors 5) Publish central dogma videos in Journal of Microbiology and Biology Education
Impacts
What was accomplished under these goals?
For Aim1, Year 2, we used the interaction between the archetype nucleotide-binding, leucine-rich-repeat (NLR) immune receptor, MLA, and the powdery mildew fungus, Blumeria graminis f. sp. hordei (Bgh), to interrogate the temporal regulation of immune signaling in barley. The plant immune system is an intricate multi-level network. Transcription factors (TFs) are essential components of the defense triggering response; after immune receptor activation, TFs drive the dynamics of gene expression and cooperatively control thousands of genes whose protein products build host defense mechanisms. TFs activate this response by physically interacting with Cis-Regulatory Elements (CREs) in the promoter regions of transcriptional targets. Activation of these networks are often mediated by NLR immune receptors. An infection-time-course transcriptome of CI 16151, carrying the Mla6 allele, and four fast-neutron-derived immune-signaling mutants, in combination with genome-wide CRE predictions and expression quantitative trait locus (eQTL) data, were used as inputs into dynGENIE3, Inferelator, and GRACE algorithms to assemble a gene regulatory network (GRN). The final GRN covered 76% of the barley genome, including 1214 transcription factors, 20125 targets, and 54698 interactions. A hierarchical structure of eight levels was found for the immune GRN. Dynamic changes in the top regulators of immunity were found, linking the MLA6 receptor with gene expression over time. Different TF families were found to regulate the response at Bgh penetration including MADS box proteins, a homeobox (HB) protein and a NAC containing protein. Other families were associated during haustorial development, such as zinc-finger, WRKY, MYB and scarecrow. Two MLA interactors are postulated to be master drivers of defense gene transcription including a homeobox and a WRKY protein. These proteins were also predicted to regulate Mla transcription through different feedback loops, suggesting a novel auto-regulatory mechanism of the immune receptor. Validation experiments are underway using ChIP-seq and ATAC-seqA For Aim #3, iPath/iTAG outreach to instruct teachers and secondary school students on the relationships between genes, alleles, phenotypes, and plant disease Goals/Deliverables at the end of the project: Multi-level case studies for biology literacy (based on discoveries from Aims 1 & 2) Undergraduate course modules (based on discoveries from Aims 1 & 2) iTAG/iPath RET whiteboard videos describing concepts in genetics and plant disease Nation-wide dissemination to underrepresented groups What was accomplished under these goals? (July 1-June 30, 2022) Multi-Level Case Study Creation: In the summer of 2021 we worked with ISU's research experiences for teachers (RET) program to identify and host a high school teaching fellow to work with Drs Wise, Peters, and Boury to develop a dilemma scenario (Jack or Jyll) based on the iTAG genotype-to-phenotype lab exercises. We are hosting this RET fellow again this year to continue the work to develop PCR tests for identified alleles that can be used to provide evidence for either side of the dilemma case study created in year 1 of the project. We have created and class-tested a case study emphasizing the biochemical pathways that connect a given genotype with its phenotype. This case study, "The great Petunia Carnage of 2017" also includes class discussion of regulations governing genetically modified organisms and public opinion of both GMO's and the regulatory process. The socioscientific issues (SSI) provide context for the course content of central dogma (DNA is copied to RNA which is the template to make a protein), Mendelian genetics, and basic biochemical principles. The manuscript for this is in preparation to be submitted to The Plant Cell. Undergraduate course modules We have created an active learning exercise (Jigsaw) teaching students about the dangers of low genetic diversity (clonal) populations in staple crops. This work provides foundational concepts of plant pathology, investigating the role of host plant on the development and progression of plant disease outbreaks and epidemics. This work was recently published at Course Source, a peer-reviewed online journal that provides teaching resources for faculty to adopt active learning in a variety of STEM disciplines. https://doi.org/10.24918/cs.2022.14 Creation of iTAG/iPath Whiteboard videos Completed: Ms. EB Wlezien has scripted whiteboard videos for several aspects of the Central Dogma of Molecular Biology, using plant species as the model system. This is particularly important, as most educational materials focus on either animal or bacterial systems. She has created the PowerPoint and recorded the first video, orienting the audience to the structure and function of DNA. In progress: This video is the first of a series that will include DNA replication, transcription, translation, and a final video connecting genotype (alleles present) to various phenotypes (e.g. disease resistance and susceptibility, 2-row-6 row, etc.) in barley. National Dissemination We have published a learning activity (Potato Late Blight and Southern corn leaf blight jigsaw) The Petunia's Case Study and GMO flipped classroom exercise have been tested and the manuscripts are in preparation for The Plant Cell and CourseSource, respectively. The whiteboard videos, when published, will be available as an Open Education Resource (OER) and be shared with colleagues at 1890 Land Grant and Hispanic Serving schools prior to their official release. Once the iTAG manual is updated, we will disseminate it using CourseSource: Evidence-based teaching resources for undergraduate biology education (https://www.coursesource.org/ ) or the American Phytopathological Society (APS) Education Resources website.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2022
Citation:
Chapman, A, JM Elmore, M McReynolds, J Walley, and RP Wise. 2022. SGT1-specific domain mutations impair interactions with the barley MLA6 immune receptor in association with loss of NLR protein. MPMI. DOI: 10.1094/MPMI-08-21-0217-R.
- Type:
Journal Articles
Status:
Published
Year Published:
2022
Citation:
Vel�squez-Zapata, V, JM Elmore, G Fuerst, RP Wise. 2022. An interolog-based barley interactome as an integration framework for NLR immune signaling. Genetics. DOI: 10.1093/genetics/iyac056.
- Type:
Journal Articles
Status:
Published
Year Published:
2022
Citation:
Wlezien EB, NT Peters, R Wise, N Boury. 2022. Role of crop genetic diversity on pathogen impact: The tale of two pathogens. CourseSource. DOI: 10.24918/cs.2022.14.
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Progress 06/01/20 to 05/31/21
Outputs
Target Audience:In our initial year, our target audience was undergraduate & graduate students and scientists at Universities, Federal Laboratories, and Industry, in addition to teachers at secondary schools. Dissemination of project results occurred through publication and presentation at National, International, and Society meetings. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?So far, we have employed 2 graduate students and one undergraduate on the project. Valeria Velásquez-Zapata is a PhD candidate in Bioinformatics & Computational Biology and heads up the informatics and gene regulation part of the project (Specific Aims 1 and 2). She is assisted by Emily Allen, an undergraduate in Biology and Science Illustration. Emily has been training in molecular biology lab protocols (DNA isolation, Polymerase chain reaction (PCR), and miscellaneous greenhouse protocols). This year, Valeria published her first (first author) paper, and presented at ISCB, SACNAS and IDEALS virtual conferences. To further her professional development, she also earned several awards. These include: Genetics Society of America Presidential Membership 2021 Schlumberger Faculty for the Future Fellowship (renewed for 3rd year) Sponsored attendance to the annual SACNAS conference Sponsored attendance to theGrad Cohort for Inclusion, Diversity, Equity, Accessibility, and Leadership Skills (IDEALS). Sponsored attendance to the CRA-W Grad Cohort for Women Workshop. ISU Graduate College Research Excellence Award 2021 (Interdepartmental Bioinformatics and Computational Biology) EB Wlezian is a Masters student in Plant Pathology & Microbiology. She is involved in Specific Aim 3: iPath/iTAG outreach to instruct teachers and secondary school students on the relationships between genes, alleles, phenotypes, and plant disease. This year, Ms. Wlezien's professional development outcomes include both procedural knowledge (e.g., how to use Qualtrics, how to grow barley plants, etc.) and conceptual knowledge on genotype and phenotypic interactions in the Informative and Spectacular Subset of the OWB barley populations and course design using backwards design principles. Greg Fuerst (USDA-ARS Biological Laboratory technician) was involved in all aspects of the project, notably as a liaison between secondary school teachers and the project team, and development and training of Y2H-NGIS sequencing protocols. How have the results been disseminated to communities of interest?This project has produced research articles in peer-reviewed journals, genome-level datasets, and novel software. The PI, Co-PIs, and students have disseminated research findings at national and international scientific meetings. Undergraduate and high school students will be mentored for future careers in STEM fields. What do you plan to do during the next reporting period to accomplish the goals?For Aim 1, we plan to develop a Gene Regulatory Network (GRN) of transcription factor (TF) - mediated control of barley and barley powdery mildew. For Aim 2, we plan to test high-confidence transcription factors and their cis-regulatory elements via ChIP Seq and gene silencing assays. For Aim 3, we expect to further develop our iPath outreach protocols, host a high school teacher for the 2021 summer, and recruit two teachers for 2022.
Impacts
What was accomplished under these goals?
Overview: Cellular dynamics are regulated by coordinated biological networks that interact at multiple levels. Functional characterization of such networks has shown that complex phenotypes are the product of profound changes in the interplay of gene and protein interactions. Our aim is to untangle the molecular properties of these networks, and thus, enable the construction of predictive models regarding the regulation of immunity in host-pathogen systems. This year, we report progress on Specific Aims 1 and 3. For Aim 1, We integrated systems biology approaches to characterize the defense of barley (Hordeum vulgare L.) to powdery mildew disease, caused by the ascomycete fungus, Blumeria graminis f. sp. hordei (Bgh). The barley MLA nucleotide-binding, leucine-rich-repeat (NLR) receptor was used as a model regulator from which to characterize the immune response. MLA represents a cross-species NLR model since its orthologs confer resistance to powdery mildew in wheat and transgenic Arabidopsis, wheat and rye Ug99 stem rust, as well as wheat stripe rust. High throughput Yeast-Two-Hybrid, Next-Generation-Interaction Screening (Y2H-NGIS) enabled identification and validation of protein-protein interactions (PPI) between MLA6 and barley targets, and these PPI were subsequently extended to additional MLA alleles. Novel interactors were positioned in a newly generated predicted barley interactome, to deduce in which cellular compartments the signaling processes localized. Data integration between the predicted interactome, transcriptome and expression quantitative trait loci (eQTL) were performed to highlight nodes, edges, structures, and modules in the signaling network. Approach and Results: To start, Y2H-SCORES, a statistical software to infer protein-protein interactions from Y2H-NGIS data, was proposed. NGIS combines yeast two-hybrid (Y2H) with deep sequencing to generate interactome networks in any organism, however, there was not a standard to provide metrics to rank high-confidence interacting proteins. Y2H-SCORES uses a Borda ensemble of three quantitative measures of Y2H-interactors, comprising: 1) an enrichment score associated with significant enrichment of prey interactors under selection, 2) a specificity score that measures the degree of interaction specificity of a prey among multi-bait comparisons, and 3) an in-frame score that ranks the selection of in-frame interactors coming from cDNA prey libraries. Using simulation, benchmarking with independent Y2H-NGIS datasets and an in-house experiment, we provided a quantitative assessment of the Y2H-SCORES performance to predict interacting partners under a wide range of experimental scenarios. These analyses helped to identify ideal experimental conditions that facilitate experimental validation including protocols such as prey library normalization, maintenance of larger culture volumes and replication of experimental treatments.Y2H-SCORES can be implemented in different yeast-based interaction screenings, with an equivalent or superior performance than existing methods. Y2H-SCORES software is available at GitHub repository https://github.com/Wiselab2/Y2H-SCORES/tree/master/Software. Second, in order to characterize the signaling networks associated with MLA-based immunity, we used interolog inference to build a protein interactome, and incorporated infection-time-course transcriptome and eQTL data. Integration of interactome and eQTL data enabled us to infer disease modules associated with two important moments in the B. graminis infection cycle, penetration and haustorial development, revealing both core and unique responses at each developmental stage. Then, using interactome and transcriptome data, we assembled resistant (R) and susceptible (S) subnetworks by selecting significant co-expressed interactions under each defense phenotype. Proteins in the differentially co-expressed (R-S) subnetwork are essential to barley immunity, connect and facilitate the flow of signaling pathways, and are linked to Mla through eQTL associations. Lastly, next-generation, yeast-two-hybrid assays identified fourteen novel MLA interactors, which were overlayed onto the interactome, predicting immune receptor localization, and signaling response over time. Our findings highlight master components of the NLR signaling cascade, linking genomic, transcriptomic, and physical interactions during the MLA-based immune response. For Aim 3, we proposed four goals for our scientific outreach: Multi-level case studies for biology literacy (based on discoveries from Aims 1 & 2) Undergraduate course modules (based on discoveries from Aims 1 & 2) iTAG/iPath RET whiteboard videos describing concepts in genetics and plant disease Nation-wide dissemination to underrepresented groups What was accomplished under these goals? We designed and deployed a gap analysis survey in Qualtrics to former RET instructors, members of the National Association of Biology teachers, and the Iowa STEM Hub regional managers. This survey identified several learning outcomes and cross-cutting concepts that are the subject of the next generation science standards (NGSS) that will be the subject of case studies. One case study will focus on HS-LS 1.1 Explain how DNA determines protein structure and function and utilize the cross-cutting concept of using models to investigation interactions. We are in the process of updating the inheritance of Traits and Genes (iTAG) lab manual to include instructions for high school and undergraduate faculty growing the Oregon Wolfe Barley (OWB). Some of these exercises are being adapted for use in general plant pathology labs. These include the use of polymerase chain reaction (PCR), a technique to amplify genes and identify allelic variation in the different mutant barley populations. We have grown the OWB mutants to make seed for use by our new and existing teacher-partners and expanded this process to include different growth conditions as a model for new faculty. While the barley growth has been optimized in the greenhouse, many high school teachers do not have access to such controlled conditions. The updates to the iTAG manual will include guidance on how to grow these plants in the classroom and solutions to potential challenges teachers may face. We have contracted with the Iowa State University Research Experience for Teachers (RET) program and have successfully identified a teacher to work with the group in the summer 2021. Melissa Greene has officially accepted the summer research experience program with our group and will work with us full time for approximately six weeks this summer. Melissa will help harvest seed for the other iTAG teachers, identifying the different phenotypes of the OWB mutants. They will use the techniques in the iTAG manual, help update the manual for use by high school and middle school teachers, and work with the educational research faculty to design short interrupted problem sets to address the NGSS standards identified in our gap analysis. Once the iTAG manual is updated, we will disseminate it using CourseSource: Evidence-based teaching resources for undergraduate biology education (https://www.coursesource.org/ )
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2021
Citation:
Vel�squez-Zapata, V, JM Elmore, S Banerjee, KS Dorman, RP Wise. 2021. Next-generation yeast-two-hybrid analysis with Y2H-SCORES identifies novel interactors of the MLA immune receptor. PLOS Computational Biology 17(4): e1008890. DOI: 10.1371/journal.pcbi.1008890
- Type:
Journal Articles
Status:
Published
Year Published:
2020
Citation:
Banerjee, S, V Vel�squez-Zapata, G Fuerst, JM Elmore, and RP Wise. 2020. NGPINT: A Next-generation protein-protein interaction software. Briefings in Bioinformatics. bbaa351. DOI: 10.1093/bib/bbaa351
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