Progress 09/01/21 to 08/31/24
Outputs
Target Audience:• College students from the SF Bay Area, mostly UC Berkeley, but also local community colleges enrolled in 2 day lab and lecture workshop on plant genomics for undergraduates • Community college and city college students in the Biotech Partners Program; disadvantaged and racial minority students getting training assistance from local biotech companies • High school students from the SF Bay Area and Southern California enrolled in 2 day lab and lecture workshop on plant genomics for high school students - • Students in the Biotech Partners Program; disadvantaged and racial minority students getting training assistance from local biotech companies • High school student internship • Undergraduate Research Apprentice Program student received research experience and mentorship • Hosting one Master's student from a wheat lab in Bari, Italy • Academic and breeders audiences reached through invited seminars and conference presentations, sitr visits and group meetings Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? We had organized a dedicated three day forward planning symposium, which provided yearly opportunities to form and discuss Individual Development Plans. Our lab symposium included structured activities to all trainees across several areas of professional development. All trainees are encouraged to fill out a yearly self-assessment using Individual Development Plans. The IDPs are further discussed in 1:1 meetings and in peer-to-peer sessions. Based on individual objectives, trainees also take additional classes and workshops. Throughout the year, our laboratory holds regular group activities. We have weekly "Laboratory meetings" to discuss research and other accomplishments and goals of each lab member. It is routine in our lab for lab members to also highlight their professional development activities in lab meetings alongside their research. We had organized a dedicated three day forward planning symposium, which provided yearly opportunities to form and discuss Individual Development Plans. Our lab symposium included structured activities to all trainees across several areas of professional development. All trainees are encouraged to fill out a yearly self-assessment using Individual Development Plans. The IDPs are further discussed in 1:1 meetings and in peer-to-peer sessions. Based on individual objectives, trainees also take additional classes and workshops. Throughout the year, our laboratory holds regular group activities. We have weekly "Laboratory meetings" to discuss research and other accomplishments and goals of each lab member. It is routine in our lab for lab members to also highlight their professional development activities in lab meetings alongside their research. How have the results been disseminated to communities of interest? Kronos genome assembly together with annotation has been publicly available on Zenodo on November 29, 2023 under the Toronto agreement. Since then, this resource has been viewed 2,000 times and has been downloaded 860 times Kronos genome has been presented to the durum sequencing consortium led by Marco Maccaferri during consortium meeting and incorporated into durum wheat long read pan-genome project EDR phenotyping, genotyping has been summarized in publication which has been preprinted on BioRxiv before journal publication Information about genomic and phenotypic resources as well as germplasm has been disseminated to multiple academic stakeholders across the US Ksenia Krasileva (PI) presented this project at multiple invited seminars. Audio and video recording of selected talks are available at lab website: https://krasilevalab.org/ What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
Goal 1: Building on the previous data, the EDR phenotyping manuscript describes stripe rust response and identification of 16 mutant lines with persistent resistance over four field trials in Davis, CA. We found that 5 of these were autoimmune lesion mimics. We used quantitative RT-PCR and found that the induction of the pathogen-responsive genes PR1, PR2 and ZLP were induced in autoimmune mutants Kr2053, Kr3339, and Kr3737 and that it was correlated with the severity of the lesion phenotype. To test the hypothesis that the persistence of stripe rust resistance may signal multifungal resistance, we collaborated with groups working with other seedling and adult stage fungal diseases. In collaboration with Meriem Aoun, Oklahoma State University, we found that autoimmune lines Kr2053 and Kr3737 are less resistant to the necrotrophic pathogen Pyrenophora tritici-repentis, causal to tan spot. In collaboration with Upinder Gill, North Dakota State University, we found that Kr2053 and Kr3737 respond to leaf rust pathogen race LrBBBQD like Kronos. However, they have increased susceptibility to mixed isolates of races LrMNPSD and LrMPPSD. These mutants do not have a resistance phenotype in assays against Oklahoma field isolates of powdery mildew. In collaboration with Xiaming Chen, USDA, we discovered that line Kr3186 has seedling resistance to PSTv-37. In collaboration with Nihdi Rawat, University of Maryland, we found that the EDR lines differed in their response to Fusarium Head Blight (FBH). 7/16 fared better at the 21 dpi timepoint and 4/16 had higher severity of FHB at 14 dpi. The FHB resistance phenotype of Kr3188 is worth investigating further. Goal 2: Previous preliminary data identified most of chromosome 1B as harboring the resistance locus for Kr620. Analysis of this data suggested that mapping efficiently would be greatly improved by having a long-read Kronos genome. We released the second version of annotation for this genome assembly in 2024 (DOI 10.5281/zenodo.11106422). Using our improved Kronos genome, we remapped the Kr620 bulked exome capture reads to confirm the interval. This data is part of our EDR manuscript under revision. We exome sequenced a different putative EDR mutant, and through this sequencing found it to be an unidentified durum line, with multifungal resistance, which we have named TD3751. This mutant was included in the trials for seedling stripe rust, tan spot, powdery mildew, and FHB. These reads are in the NCBI SRA but have not yet been released. We grew a Kronos x TD3751 F2 population, and sequenced bulked pools for mapping which identified a 300Mb region of chromosome 1BS. We identified recombinants in the population and used the Kronos genome and the TD3751 exome data to design new molecular markers. Fine-mapping is ongoing. Goal 3: To modulate the wheat immunity, we have focused on the promoters involved in pathogen response and performed their engineering to validate their responses. We also generated RNA-seq data to mine stress-responsive genes. We analyzed the transcriptomic data from previous studies to identify up-regulated genes under stripe rust infection. From this analysis, we selected three candidate genes (PR1, PR2 and ZLP) and utilized their promoter to express the RUBY gene in Kronos and autoimmune lines. Preliminary analysis of PR1:RUBY transgenics showed the induction of plant immune response under stripe rust infection. We have generated RNA-seq data from Kronos by subjecting stripe rust with or without drought and salinity stress. This data will be helpful to identify common stress-responsive pathways under biotic and abiotic stresses. We have generated RNA-seq data from the calli of Kronos infected with or without Agrobacterium from callus induction and regeneration stages. This data will help identify stage and tissue-specific expressing genes. Utilizing these genes' specific promoters would be useful in restricting the expression of target transgenes within the specific plant cell developmental stage, for instance, the expression of CRISPR-Cas, to avoid unnecessary molecular burden for later plant developmental stages.
Publications
- Type:
Peer Reviewed Journal Articles
Status:
Accepted
Year Published:
2024
Citation:
C Lunde, K Seong, R Kumar, A Deatker, B Chhabra, M Wang, S Kaur, Song S, Palayur A, Davies C, Cumberlich W, Gill U, Rawat N, Chen X, Aoun M, Mundt C, Krasileva K (2024) Wheat Enhanced Disease Resistance EMS-Mutants Include Lesion-mimics With Adult Plant Resistance to Stripe Rust Biorxiv doi: https://doi.org/10.1101/2024.05.10.593581
accepted pending revision at Molecular Breeding
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Progress 09/01/22 to 08/31/23
Outputs
Target Audience: College students from the SF Bay Area, mostly UC Berkeley, but also local community colleges enrolled in 2 day lab and lecture workshop on plant genomics for undergraduates Community college and city college students in the Biotech Partners Program; disadvantaged and racial minority students getting training assistance from local biotech companies High school students from the SF Bay Area and Southern California enrolled in 2 day lab and lecture workshop on plant genomics for high school students - Students in the Biotech Partners Program; disadvantaged and racial minority students getting training assistance from local biotech companies High school student internship Undergraduate Research Apprentice Program student received research experience and mentorship Hosting one Master's student from a wheat lab in Bari, Italy Academic and breeders audiences reached through invited seminars and conference presentations Changes/Problems: One previously identified priority mutant, showing high levels of stripe rust resistance and septoria resistance was sent for exome capture (as had not been included in previous experiments). Analyses of this mutant line compared to Kronos showed a seed admixture. We are in the process of checking for known disease resistance genes in the mapped interval on 1BS and following up if we find it to be a source of novel (uncloned) resistance. It is unique in that it has both seedling and adult stripe rust resistance. The DNA Technologies Core at UC Davis was unable to get HMW gDNA from Kronos so we were delayed about 6 months troubleshooting this ourselves, however, we now successfully completed this goal and generated a chromosome level assembly. What opportunities for training and professional development has the project provided?All project participants: We had organized a dedicated three day forward planning symposium, which provided yearly opportunities to form and discuss Individual Development Plans. Our lab symposium included structured activities to all trainees across several areas of professional development. Examples of activities from the past year included "Research Forward Planning and Project Management", "Science Communication to wide audience and Improving Presentations for Scientific Audience", "Ethical use of ChatGPT and other AI technologies in research, teaching and outreach", "Career Development'', "Mentorship". All trainees are encouraged to fill out a yearly self-assessment using Individual Development Plans. The IDPs are further discussed in 1:1 meetings and in peer-to-peer sessions. Based on individual objectives, trainees also take additional classes and workshops. Throughout the year, our laboratory holds regular group activities. We have weekly "Laboratory meetings" to discuss research and other accomplishments and goals of each lab member. It is routine in our lab for lab members to also highlight their professional development activities in lab meetings alongside their research. The lab holds several "Journal clubs" oriented towards introducing new trainees to the field or covering and discussing literature at a particular focus topic as well as "Coding Club" to facilitate knowledge exchange and peer to peer training in bioinformatics and data visualization. Last year we also started "Writing club" to facilitate scientific writing skill development and exchange. Individual participant training and professional development: Research Specialist (China Shaw) and postdoc (Rakesh Kumar) attended the American Phytopathological Society Meeting in Denver, CO from August 12 - 16, 2023. They met with PIs or representatives from collaborating laboratories including: Mundt Lab (Oregon State University), Aoun Lab (Oklahoma State University), Rawat Lab (University of Maryland, DE). They also initiated a new collaboration with Meinan Wang and Xianmen Chen of Washington State University and USDA, Pullman to assess seedling response of EDR lines to PSTv-37 inoculation. China took implicit bias training and served on a hiring committee for a senior greenhouse technician. Rakesh (Postdoc) attended a 2023 Summer CCB Python Bootcamp organized by UC Berkeley from June 12-16, 2023 Rakesh (Postdoc) participated in a Scientific writing workshop organized by Innovative Genomics Institutes, UC Berkeley on Oct 11, 2023 Undergraduate, Ann Palayur, UC Berkeley, earned credits as a URAP (Undergraduate Research Apprentice) characterizing cold enhancement of autoimmune EDR mutants and learning tissue culture to support the transgenic experiments International visitor scholar (Six-month research program): Giuseppe, University of Bari Aldo Moro, Italy will characterize rust-inducible promoters August, 2023 to February, 2024 toward completion of a Master's Degree at his home institution. How have the results been disseminated to communities of interest? Rakesh (Postdoc) presented research updates at Plant and Animal Genomics (PAG30) in San Diego, CA from Jan 12-17, 2023 China (Research Specialist) and Rakesh (postdoc) presented posters at the American Phytopathological Society Meeting in Denver, CO from August 12 - 16, 2023. Ksenia Krasileva (PI) presented this project at multiple invited seminars. Audio and video recording of selected talks are available at lab website: https://krasilevalab.org/ What do you plan to do during the next reporting period to accomplish the goals?Goal 1 We will use PR1::RUBY Kr2053 and Kr3737 transformants to look at early lesion formation in autoimmune lines that have distinct phenotypes: small ubiquitous lesions and medium lesions that form mostly at the leaf centers and tips, respectively. We will evaluate these lines with and without pathogens and with and without cold stress. These lines will allow us to conduct spatial transcriptomics to provide insight into temperature controlled enhanced resistance inducible gene expression that is site-specific to HR responses in wheat. We will summarize all EDR mutant phenotypic observations including: field and controlled inoculation data of mutant lines at seedling and adult stages, our rationale for prioritizing them by their stripe rust and multi-pathogen resistances as well as listing their susceptibility (pathogen tradeoff) and yield data and make it available through publication as a phenotypic mutant survey paper. Goal 2 Kronos x Kr2876 F2 plants are growing in a rust nursery in Davis, CA. In May/June 2023, these will be scored for resistance/susceptibility to stripe rust and individual gDNA samples will be taken to make bulked R and S samples for mapping. We will grow plants to maturity to generate homozygous R and S lines.To reduce the potentially large number of gene candidates in our mapping interval, we will grow F3 lines in growth chambers with and without rust inoculation, and collect tissue samples for RNA for expression analysis to prioritize genes induced under pathogen infection. For line *3751, we will use scored individual F2 gDNA samples taken from the field to find recombinants and eliminate as possibilities or further fine-map the two mapped (but uncloned) Yr genes in the 1BS *3751 mapping interval, Yr64 and Yr65, using existing published markers. We harvested F3 heads and will grow F3 lines in growth chambers with and without rust inoculation, and collect tissue samples for RNA for expression analysis to prioritize genes induced under pathogen infection. We will characterize 3751* x Kr244 double mutants for response to stripe rust by growing them in the rust nursery in Davis We will produce annotations of Kronos genome and release it to the community prior to publication. We are planning to publish it together with comparative genomic analyses of disease resistance genes across wheat lines. Goal 3 We will finish generating stable transgenic lines with 3 additional promoter reporters. We will use these promoters together with PR1 for inducible disease resistance expression. We will validate dnd1 gene-edited lines and select lines with single, double, and triple-edited copies. Together with our collaborators, we will phenotype these lines against major wheat pathogens.
Impacts
What was accomplished under these goals?
Goal 1: Phenotype gain-of-resistance wheat mutants, analyze effects of resistance on yield and its interaction with the environment. In this reporting period, we continued to characterize autoimmune mutant lines and identified those inducible under colder temperatures. A subset of autoimmune mutants grown in growth chambers at 16C/10C day/night showed cold-temperature inducible lesion formation compared to sibling controls grown at 18C/11C day/night. We have generated stable transgenic lines with PR1::RUBY reporter and observed inducible expression of RUBY surrounding lesions, confirming gene inducible auto-immune phenotype. We completed field observations of stripe rust and a thousand kernel weight yield assessment of gain-of-resistance wheat mutants. Though several national collaborations, have gathered disease resistance phenotype for Kronos and selected gain-of-resistance lines for five major wheat pathogens: septoria tritici blotch, fusarium head blight, leaf rust (races BBBQD, MNPSD, and MPPSD), tan spot US race 1, and powdery mildew (Oklahoma field collection). We used combined resistance data to make a revised prioritized mutant list with multi-pathogen resistance. Goal 2: Identify mutations responsible for increased resistance to stripe rust. During this reporting period, we phenotyped two mapping populations and performed mapping by sequencing for the priority mutant line. While we were able to map causative phenotype to chromosome 1BS, we identified that this mutant is likely an admixture in the breeder's original seed. Since the resistance is broad spectrum, lacks HR or other phenotypes associated with known Yr genes, we plan to follow up these highly resistant lines, first checking for presence of cloned genes. For the rest of the mutants, we prioritized two lines and collected samples for the top highly resistant autoimmune line without yield penalty as measured by TKW in a rust infected field and compared to Kronos. We identified an additional priority line for mapping during next season. Importantly, we leveraged long read PacBio HiFi sequencing technology to produce chromosome level assembly of Kronos. We optimized high molecular weight DNA extraction from wheat, which presented several challenges, and produced 526 Gbp of high-fidelity (HiFi) reads corresponding to 50X of the genomic coverage. providing sufficient information to assemble a high-quality reference genome for Kronos. Combined with chromosome conformation capture sequencing (Hi-C) data that inform genomic contact and proximity, we recovered 14 chromosome level scaffolds, 8 of which spanned across both telomeres. Kronos is a founder line for the widely used reverse genetics resource in wheat (exome-capture sequenced TILLING population, Krasileva et al PNAS 2018, cited 456 times), therefore, our high quality genome is of significant interest to both breeders and scientific community. The contigs and scaffolds are made publicly available through Zenodo: https://doi.org/10.5281/zenodo.10215402. We plan to release the genome annotation within this year. ?We have also established a high efficiency transformation of Kronos within our lab. Goal 3: Modulate activation of immunity in wheat using natural and synthetic promoters. During this reporting period, we prioritized three inducible promoters and cloned them for transformation into wild type and autoimmune Kronos lines. We have also started to generate Kronos lines with edited enhanced disease resistance gene candidate Defense No Death 1 (DND1) previously identified in Arabidopsis. DND1 encodes a Calcium channel (Clough et al. 2000); Kronos has three copies of the gene. We have targeted all three copies with 2 guide RNAs to generate both individual and multi-gene knockouts. We are propagating these plants to produce homozygous recessive mutant plants to assess the effect of dnd1 mutations under pathogen infection.
Publications
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2023
Citation:
Rakesh Kumar, China Lunde, Kyungyong Seong, Joshua Hegarty, Sarah Song, Chris Mundt, Ksenia Krasileva - "Identification and engineering of broad-spectrum enhanced disease resistance alleles in wheat" - APS Plant Health, Denver, Colorado, USA. August 12-16, 2023, poster
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2023
Citation:
Rakesh Kumar, China Lunde, Joshua Hegarty, Sarah Song, Chris Mundt, Ksenia Krasileva - "Exploring Genetic Elements and Strategies for the Improvement of Rust Resistance in Wheat" - The Plant and Animal Genomics, Translational Genomics Workshop, San Diego, CA, USA. January 14th 2023, invited talk
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2023
Citation:
China Lunde, Chris Mundt, Alex Schultink, Rakesh Kumar, Kyungyong Seong, Joshua Hegarty, Jorge Dubcovsky, Ksenia Krasileva - "Strategies and resources for improving durum wheat Kronos - for stripe rust resistance and other traits"- APS Plant Health, August 12 - 16, 2023, poster
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2023
Citation:
Ksenia Krasileva - "From Natural Diversity to Engineering: Rational Optimization of Wheat Disease Resistance Genes" - The Plant and Animal Genomics, New Approaches for Developing Disease Resistance in Cereals Workshop, January 14th 2023, invited talk
- Type:
Other
Status:
Published
Year Published:
2023
Citation:
The contigs and scaffolds are made publicly available through Zenodo: https://doi.org/10.5281/zenodo.10215402. We plan to release the genome annotation within this year.
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Progress 09/01/21 to 08/31/22
Outputs
Target Audience: High School students - reached by teaching activities in our Genomes and Plant Health workshop described in 'other products/outputs' Undergraduate students - reached by teaching activities in our Genomes and Plant Health workshop described in 'other products/outputs' High School teachers - reached through discussion of genomics and bioinformatics curriculum with Biotech Partners (http://www.biotechpartners.org) Academic researchers and members of seed companies - reached through conference presentations, invited seminars, virtual zoom meetings and online discussions. 2022. PI has been consulting and invited to serve on the advisory board for development of new curricula in Genomics and Computational Biology for High School students with Biotech Partners (http://www.biotechpartners.org) 2021, 2022. Under this grant, we developed a new summer workshop "Genomics and Plant Health" https://krasilevalab.org/outreach/ This workshop has been delivered to Undergraduate research students (2x). High school students (2x). Together with graduate students and postdocs, we developed new teaching and learning material on following topics "Decoding Genomes", "Genome assembly and Annotation", "Transposable Elements", "Protein structures", "Genome Editing" and "Plant Microbiomes". Teaching materials included lectures, demonstrations (Oxford Nanopore Sequencing on wheat DNA sample), hands on computational biology exercises using web resources and Google Colab to demystify genomics, spur interest in data science and engage students from very diverse backgrounds therefore contributing to removing barriers for historically underrepresented groups in data sciences. PI has a professional Twitter account highlighting project updates and related material to over 5,000 community members. Project has been highlighted on the lab website, which includes both project descriptions and posted recordings of the talks. According to google analytics, the website had 5,800 views over the reporting period. https://krasilevalab.org/ Changes/Problems:We have continued to experience some challenges linked to COVID-19 pandemic, including interrupted experiments in the lab due to COVID safety precautions with lab members being exposed to COVID or being sick, delayed ordering and deliveries. We have also had a longer time with delivery and installation of our inoculation growth chamber which did not allow us to do controlled inoculation experiments and microscopy in year 1 as was proposed. We have also decided to adopt new HiFi error free sequencing of Kronos genome that has proven over the past year to produce gold standard high quality genome assembly in place of more error prone Oxford Nanopore. Despite all limitations and minor adjustments in methodologies that led to delays, ourgoals for the project remain unchanged. What opportunities for training and professional development has the project provided?Each year, our laboratory organizes a dedicated three day forward planning symposium, which provides yearly opportunities to form and discuss Individual Development Plans. We provide structured activities to all lab members across several areas of professional development. Examples of activities from the past year included "Research Forward Planning and Project Management", "Science Communication to wide audience and Improving Presentations for Scientific Audience", "Career Development'', "Mentorship". Throughout the year, we have regular meetings and additional opportunities for professional development described below: Graduate students and postdocs are encouraged to fill out a yearly self-assessment using Individual Development Plans. The IDPs are further discussed in 1:1 meetings and in peer-to-peer sessions. Based on individual objectives, trainees also take additional classes and workshops.? Throughout the year, our laboratory holds regular group activities. We have weekly "Laboratory meetings" to discuss research and other accomplishments and goals of each lab member. It is routine in our lab for lab members to also highlight their professional development activities in lab meetings alongside their research. The lab holds several "Journal clubs" oriented towards introducing undergraduates and new lab members to the field or covering and discussing literature at a particular focus topic, "Coding Club" to facilitate knowledge exchange and peer to peer training in bioinformatics and data visualization and "Writing club" for maintaining and developing scientific writing skills. PI sets aside an hour a week for meeting with each graduate student, postdoc and professional. Each lab member meets with the PI at least once every two weeks for an hour and most trainees choose to meet weekly. Daily, we interact on Slack with dedicated channels to literature discussions, conferences and meetings, bioinformatics, protocols as well as specific project discussions. Graduate students, postdocs and professionals involved in the project are encouraged to attend at least one national and one international conference a year and present their research as talks or posters. Most conferences for the past year were still remote due to the pandemic. Despite that, Specialist China Lunde Shaw presented a poster April 2022 in St. Louis at the 60th Maize Genetics Conference describing the wheat EDR mutants and including work by a graduate student mentee, also an author on the poster and attended the Borlaug Global Rust Initiative Technical Workshop. All project members have numerous opportunities each week to attend seminars across campus. To facilitate professional network development, PI invited trainees to 1:1 meeting with seminar speakers. The project provided both bioinformatics and molecular biology training as well as experience working with wheat and genetics to a graduate student, an undergraduate researcher and a high school student. Project participants also had opportunities for leadership development and training others. For the past year, members of the project had the opportunity to lead hands-on our "genomes and Plant Health" workshop for high school students and undergraduates using their research data and teaching material that they developed. How have the results been disseminated to communities of interest?Results have been disseminated to scientific communities through conference presentations, invited talks and workshops. Specialist China Lunde Shaw presented a poster April 2022 in St. Louis at the 60th Maize Genetics Conference describing the wheat EDR mutants and attended the Borlaug Global Rust Initiative Technical Workshop, September 2022. Ksenia Krasileva presented the project at the American Phytopathology Society as well as in multiple invited seminars. Recordings of several conferences talks and seminars as well as teaching materials are available on our website: https://krasilevalab.org/ What do you plan to do during the next reporting period to accomplish the goals?Goal 1: Phenotype gain-of-resistance wheat mutants, analyze effects of resistance on yield and its interaction with the environment. Continue to collect field and controlled chamber conditions phenotypic data using multiple pathogens and in collaboration with partners described above. Perform RNA-sequencing on tissue collected from lesion mimic mutants under cold treatment conditions described above compared to the world type control. Generate crosses between reporter lines pPR1::eGFP. Transgene positive plants were bulked for crosses with selected lesion mimic EDR lines Perform abiotic stress heat tests on selected lines in controlled conditions and evaluate resistance - heat stress tradeoffs. Optimize rust inoculation in our growth chamber, once successful begin to assess when in the infection process the pathogen is stopped in EDR mutants Goal 2: Identify mutations responsible for increased resistance to stripe rust. Generate genome assembly and annotation of the Triticum turgidum Kronos using PacBio HiFi sequencing. Reanalyze existing data on updated genome assembly and develop CAPS markers for validating co-segregating regions. Prioritize 'green' EDR lines that showed enhanced resistance against multiple pathogens, including both rust and Septoria for gene cloning by phenotyping during rust field season in Davis in April 2023 followed by bulk segregant analysis. Goal 3: Modulate activation of immunity in wheat using natural and synthetic promoters. Synthesize several pre-selected pathogen inducible promoters and transform them into Kronos and selected EDR backgrounds with ruby reporter for evaluation under rust infection.
Impacts
What was accomplished under these goals?
Goal 1: Phenotype gain-of-resistance wheat mutants, analyze effects of resistance on yield and its interaction with the environment. Last year we added a 3rd year field observations in Davis for both rust infection and yield measurements of the enhanced disease resistance (EDR) mutant lines. In total, 33 lines were evaluated, and 32 continued to show enhanced disease resistance in the field. Among the lines, we continued to observe a variety of cell death responses, from two lines being fully 'green' and having no cell death to extensive cell death that was visible even before pathogen infection. We have previously observed that some enhanced disease resistance (EDR) mutant lines showed HR/cell death before pathogen infection. Based on similar phenotypes described in other species, we called these lines "lesion mimic". We hypothesized that lesion mimic wheat lines had enhanced pathogenesis related gene expression and might also be temperature sensitive. In the past year, we successfully tested both of these hypotheses by growing 8 of these lines in two different temperature conditions and in a controlled environment with no pathogen infection and conducting qPCR analyses of pathogenesis related (PR) gene expression. At 22°C day/18°C night temperature regime, in a growth chamber, no lesions were visible, however lower 16°C day/10°C night temperatures resulted in lesion formation in 4 of 5 EDR lines tested, 2 had expressive phenotypes and were chosen for follow-up transcriptional analysis. Pr1 gene expression was measured at three developmental timepoints in cold-sensitive autoimmune EDR lines. Interestingly, Pr1 was upregulated in one of the lesion mimic lines but not in the other. This suggests that Pr1 induction is not necessary to induce HR and yet may allow a criteria by which to sort the EDR lines into their affected pathways. Samples were simultaneously collected for RNA-Sequencing and will be processed now that the cold treatment response has been verified. A high-throughput image analysis protocol to evaluate thousand kernel weights (TKW) was developed as an undergraduate research project by Sarah Song; data has been collected thus far with the result that lines showing EDR response, including some of 'lesion mimic' lines show similar or enhanced yield compared to wild type control when grown in the rust-infected field. To address whether our EDR lines would have broad spectrum resistance or any trade offs against other pathogens, we continued collaboration with Dr Chris Mundt (Oregon State University). We have received phenotypic data for Septoria for 31 lines, showing the two 'green' lines along 6 others showed enhanced resistance to Septoria, relative to Kronos which is moderately resistant (having a subjective score between the moderate and susceptible checks, Madsen and Stephens, respectively). The 'lesion mimic' lines showed enhanced susceptibility to Septoria showing the tradeoff between activation of cells-death response and immunity to a necrotrophic pathogen. Presentation of this project at the Annual Phytopathology Society led to another collaboration with Dr. Upinder Gill at North Dakota State University and we have distributed seeds of key EDR lines for evaluation against leaf rust. Dr. Meriem Aoun at Oklahoma State University is testing the response of Kronos to several pathogens: leaf rust, powdery mildew, spot blotch, tan spot, wheat soil borne mosaic virus, barley yellow dwarf virus. Kronos was found to be moderately resistant to spot blotch and moderately susceptible to tan spot. Other results are pending. We have also received evaluation of EDR lines against Fusarium Head Blight from collaboration with Dr Nidhi Rawat at the University of Maryland indicating diversity in response to FHB in the EDR lines in the field with follow up experiments in progress, focussing on the 3 lines that fared best in the pilot field trial, plus the 2 green and 2 autoimmune lines of particular interest.. We advanced generation of reporter lines pPR1::eGFP. Transgene positive plants were bulked for crosses with selected lesion mimic EDR lines with confirmed upregulated PR1 expression. To start testing at which point during the infection cycle the pathogen is stopped, we purchased and installed a rust-dedicated growth chamber, inoculation protocols are under deployment. Goal 2: Identify mutations responsible for increased resistance to stripe rust. For 3 lines, we have analyzed exome capture data on resistant and susceptible bulks in collaboration with Dr Alex Schultink (Fortiphyte), this yielded several genomic regions with high mutation frequencies associated with EDR. These regions contain disease resistance gene clusters, which strengthened the need for a contiguous T. turgidum Kronos genome. We have evaluated several HMW genomic DNA extraction methods, sequencing options and sequencing providers and have initiated a project with UC Davis Genome sequencing center to produce 30X PAcBio HiFi coverage of Kronos genome. Based on most up to date studies, we expect this will achieve chromosome level genome assembly. We already have RNAseq data for Kronos from PI's postdoc work for annotation. During the past year, we also established bioinformatic genome annotation pipelines in the lab that were applied to a collaborative tomato genome project. Using the cold growth chamber phenotyping of one unusual EDR line with a chlorotic phenotype but no lesions, single F2 heads were collected from chlorotic segregants and wildtype sibs yielding BC1F3 plants for inclusion in further GBS/BSA studies described above. Crosses collected at Davis in June 2022 included F3 heads of double mutant lines between the EDR lines with 'very green' phenotypes and double mutants between an EDR line with coalescing lesions and one with sporadic lesions. Although analysis of epistasis is complicated by the dominant nature of these mutations, by combining the defects we may learn about the nature of the processes contributing to resistance. We also collected more BC1F4 heads of one of the green lines to generate more informative recombinants for our GBS pipeline. To validate any segregating mutations, we have compared KASP and CAPS protocols based on marker data from our analyzed lines described above and settled on CAPS as the most efficient, high throughput and robust method to take forward. Given polyploid nature of the wheat genome, unavailability of Kronos reference and complex nature of disease resistance gene families, we are still optimizing marker design and will likely use a combination of methods. Goal 3: Modulate activation of immunity in wheat using natural and synthetic promoters. We have conducted large scale analyses of promoter elements induced by wheat pathogens. We filtered datasets for genes that showed enhanced expression only after rust exposure and not septoria and powdery mildew or the drought and heat stress, which resulted in a total of 200 transcripts. We extracted up to 2000 bp upstream of these genes and performed motif enrichment analysis, which uncovered 9 statistically enriched motifs. Based on expression changes and motifs, we selected 12 candidate promoters for further investigation. A postdoc hired on this project in the past year who has extensive tissue culture experience initiated construct designs for in lab transformation of synthetic constructs into Kronos and EDR lines. He has already designed and synthesized two vector based transformation systems with Ruby reporter that would allow us to easily test selected promoters in vivo with and without rust inoculations in controlled growth chamber environments in wild type and EDR backgrounds.
Publications
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