Progress 05/01/23 to 04/30/24
Outputs
Target Audience:
Nothing Reported
Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?The project supported two graduate students, Kaylie Richard and Taylor Ziccardi, who received interdisciplinary trainingin biomolecular engineering and plant biology/pathology. Ms. Richard is a fourth-year PhD student who has been developing the AlphaFold computational pipeline and testing the inducible promoters as described above. Ms. Ziccardi is a first-year PhD student who is learning some of the basic molecular biology techniques needed for the project. The project also supported a research scientist, Dr. LindaJeanguenin, who has led the optimization of nuclei isolation and snRNA-seq. Furthermore, Ms. Richard has been mentoring two undergraduate researchers and Dr. Jeanguenin has been mentoringtwo undergraduate researchers. How have the results been disseminated to communities of interest?The results have been disseminated in three published journal articles, a conference oral presentation, and a conference poster presentation (all described under Products). What do you plan to do during the next reporting period to accomplish the goals?In alignment with the first goal, we will continue to optimize the signal-to-noise ratio for specific LeEIX2/EIX binding in ribosome display, but we have identified XEG1/RXEG1 as a potentially more promising pair for specificity engineering using both AlphaFold and ribosome display. Inalignment with the second goal, we will analyze the results of our single-nucleus RNAseq data to identify novel promoters that can link PTI to a nuclear fluorescent reporter readout. Then, we will demonstrate proof-of-principle of our high-throughput selection system and move on to library selections.
Impacts
What was accomplished under these goals?
In alignment with the first goal, we have begun to develop a computational pipeline using AlphaFold to better identify putative binding interactions between LRR proteins and their native ligands. However, most of the work in the past year has been aligned with the second goal of the project. While pattern recognition receptors (PRRs) are major determinants of immune specificities to pathogens, the search for PRRs with new recognition specificities can be time consuming, limiting the ability to engineer plants against rapidly evolving pathogens. To characterize the function of PRRs in a more high-throughput way, we are developing single-nuclei reporter systems in model plants that will allow the function of a PRR in a single cell to be quantified and sequenced to identify the corresponding protein sequence. Previously, we identified a histone-tagged mNeonGreen (HTA6-mNG) fusion protein that localizes to and is retained in the nucleus after nuclei isolation, allowing for characterization with flow cytometry. Next, we evaluated promoters that would enable the specific expression of HTA6-mNG only when pattern-triggered immunity (PTI) is activated, not under general stress response. LysI from N. benthamiana has previously been reported as an PTI marker gene in the context of Agrobacterium transient expressions. Using Agrobacterium-mediated transformation in N. benthamiana leaf tissue, we measured the expression of HTA6-mNG under the control of the LysI promoter in addition to three other candidate promoters from A. thaliana in response to flg22, a peptide from bacterial flagellin that induces PTI. While HTA6-mNG was expressed under flg22 infiltration, the reporter was also expressed in the mock infiltration conditions, indicating that these promoters either have high basal levels of expression or are responding to the Agrobacterium. We also observed no substantial differences in transcription levels under flg22 versus mock infiltration conditions for these inducible promoters in the context of transient Agrobacterium infiltrations. Since the A. thaliana promoters will likely behave differently in N. benthamiana compared to the native host, we are currently in the process of testing these promoters in stable A. thaliana lines. We are also performing an RNA-seq time-course experiment to identify additional promoter candidates that are upregulated by flg22 but not Agrobacterium in N. benthamiana. In parallel, we worked on optimization of nuclear preparation from Arabidopsis leaves, including using isolated nuclei for single-nucleus (sn) RNA-seq. Nuclear preparation from green plant tissue is challenging as green plant tissue has so many chloroplasts (~50 times of nuclei) and the sizes of the chloroplasts and nuclei are similar. Conventionally, high-concentration detergents are used to remove chloroplasts, which are more sensitive to detergents than nuclei. However, detergents also damage nuclei to some extent. For the purpose of snRNA-seq, damaged nuclei leak RNA and lose representation in the snRNA-seq data, which could bias the data toward more sturdy nuclei. We plan to use PIPseq technology instead of commonly used 10xGenomics Chromium technology. PIPseq is much cheaper per sample (at most 1/5 of the cost of Chromium per sample). We ran a pilot PIPseq experiment with Arabidopsis nuclear preps and found that PIPseq does not require removal of chloroplasts and allows use of fixed nuclei. These two characteristics of PIPseq are expected to substantially reduce the bias among nuclear populations. Although the UMI counts per nucleus obtained with PIPseq were about half of that by 10xGenomics Chromium, the quality and less-biased nature of the PIPseq data is preferable for our interest in collective dynamic behaviors of transcripts of thousands of genes in each single cell.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2023
Citation:
Rachel A Hillmer, Daisuke Igarashi, Thomas Stoddard, You Lu, Xiaotong Liu, Kenichi Tsuda, and Fumiaki Katagiri. The Kinetics and Basal Levels of the Transcriptome Response During Effector-Triggered Immunity in Arabidopsis are mainly controlled by Four Immune Signaling Sectors. Journal of Bioinformatics and Systems Biology 6 (2023): 347-363. doi:10.26502/jbsb.5107070
- Type:
Journal Articles
Status:
Published
Year Published:
2024
Citation:
Xiaotong Liu, Daisuke Igarashi, Rachel A Hillmer, Thomas Stoddard, You Lu, Kenichi Tsuda, Chad L Myers, and Fumiaki Katagiri. Decomposition of dynamic transcriptomic responses during effector-triggered immunity reveals conserved responses in two distinct plant cell populations. Plant Communications 5 (2024): 100882. doi: 10.1016/j.xplc.2024.100882
- Type:
Journal Articles
Status:
Published
Year Published:
2024
Citation:
Fumiaki Katagiri. An averaging model for analysis and interpretation of high-order genetic interactions. PLoS One 19 (2024): e0299525. doi: 10.1371/journal.pone.0299525
- Type:
Conference Papers and Presentations
Status:
Accepted
Year Published:
2024
Citation:
Kaylie Richard. Single-cell level detection of pattern-triggered immunity activation. 2024 Plant Molecular Biology Gordon Research Seminar.
- Type:
Conference Papers and Presentations
Status:
Accepted
Year Published:
2024
Citation:
Kaylie Richard, Linda Jeanguenin, Casim Sarkar, and Fumiaki Katagiri. Single-cell level detection of pattern-triggered immunity activation. 2024 Plant Molecular Biology Gordon Research Conference.
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Progress 05/01/22 to 04/30/23
Outputs
Target Audience:
Nothing Reported
Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?The project supported two graduate students, Kaylie Richard and Andrew Baldys, who received interdisciplinary training inbiomolecular engineering and plant biology/pathology. Ms. Richard is a third-year PhD student who has been advancing the new selection strategies described above. Mr. Baldys was making good progress as well, but chose to leave his doctoral program during the past reporting period for personal reasons.The project also supporteda postdoctoral associate, Dr. Linda Jeanguenin, who provided expertise to optimize receptor expression and downstream signaling/transcriptome analysis in plants. Furthermore, Ms. Richard has been mentoring one undergraduate researcher and Dr. Jeanguenin has been mentoring three undergraduate researchers. How have the results been disseminated to communities of interest?
Nothing Reported
What do you plan to do during the next reporting period to accomplish the goals?In alignment with the first goal, we will develop and test protein libraries for ribosome display that diversify the newly identifiedloop regions in LeEIX2 to better understand binding to EIX and to alter the specificity of LeEIX2to new targets. In alignment with the second goal, we will combine our inducible reporter system withCRISPR-Cas9 mediated site-directed mutagenesis of FLS2 to enable fluorescence-activated nuclei sorting and recovery of FLS2 variants that recognize flg22 variants that evade wild-type FLS2 recognition.
Impacts
What was accomplished under these goals?
We have made progress on both major goals of the project: In alignment with the first goal, we have combined computational structural predictions with cell-free protein display methods to engineer the binding specificity of plant immune receptors. As a case study, we focused on the receptor-like protein (RLP) LeEIX2 from Solanum lycopersicum, which recognizes ethylene inducing xylanase (EIX) from the fungus Trichoderma viride. To better understand the interaction between LeEIX2 and EIX, we modeled the complex formed by EIX and the LeEIX2 ectodomain using AlphaFold2-Multimer. In addition to the two EIX-binding hotspots located in the LRR core previously identified by our lab, two loop regions of LeEIX2 are predicted to interact with the catalytic site of EIX. To further interrogate the role of the loop regions in EIX recognition, we examined the sequence conservation of the loops relative to the surrounding LRR region. The N-terminal loopand the first island domain's beta sheet-loop-beta sheet show higher sequence diversity compared to the surrounding N-terminal capping region and the next four LRR units, respectively. By comparing these structures to the crystal structure of a similar receptor, RXEG1, and its target XEG1 xyloglucanase, we hypothesize that increased diversity corresponds to the diversity of targets recognized by this class of receptor-like proteins. In alignment with the second goal, we designed a reporter method to link pattern recognition receptor(PRR) immune activation to a detectable fluorescent output, with activation of a transmembrane PRR by a pathogen associated protein induces the expression of a nuclear-localized fluorescent protein. For proof-of-concept, we focused on the activation of FLS2, a transmembrane RLK, by the bacterial flagellin peptide flg22. Previously published RNA-seq data was used to identify three genes with increased expression after fgl22 but not mock infiltrations. The promoters of these genes will be tested in planta to measure the expression of the fluorescent reporter after flg22 and mock infiltration and identify the promoter with the highest signal to noise ratio. To design the fluorescent reporter protein, we tested the following fusion proteins under constitutive expression: 3xmVenus, mNeonGreen (mNG), mNG-β-glucuronidase (Gus), mNG (2x)-Gus, and histone H2A-mNG. Only the histone-mNG fusion resulted in isolated fluorescent nuclei confirmed by flow cytometry and fluorescence microscopy.
Publications
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2023
Citation:
Katagiri, F. How might highly resilient plant immunity have evolved? A "multiverse" hypothesis. Physiological and Molecular Plant Pathology 124, 101968 (2023). https://doi.org/10.1016/j.pmpp.2023.101968
- Type:
Other
Status:
Other
Year Published:
2022
Citation:
Katagiri, F. An averaging model for analysis and interpretation of high-order genetic interactions. bioRxiv (2022). https://doi.org/10.1101/2022.02.21.481332
- Type:
Other
Status:
Other
Year Published:
2023
Citation:
Hillmer R.A., Igarashi, D., Stoddard, T., Lu, Y., Liu, X., Tsuda, K., Katagiri, F. The kinetics and basal levels of the transcriptome response during Effector-Triggered Immunity in Arabidopsis are mainly controlled by four immune signaling sectors. bioRxiv (2023). https://doi.org/10.1101/2023.05.10.540266
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Progress 05/01/21 to 04/30/22
Outputs
Target Audience:
Nothing Reported
Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?The project supported two graduate students, Kaylie Richardand Andrew Baldys, who received interdisciplinary training in biomolecular engineering and plant biology/pathology. Ms. Richard is a second-year PhD student who has been advancing a new selection strategy that was initiated by a previous PhD student who was supported by this grant (now graduated). Mr. Baldys is a second-year PhD student who has been learning some of the basic molecular engineering experimental tools, like ribosome display. The project also supported a postdoctoral associate, Dr. Anshulika Rai, who provided expertise to test receptor expression and hypersensitive responses in plants. How have the results been disseminated to communities of interest?
Nothing Reported
What do you plan to do during the next reporting period to accomplish the goals?We have two primary goals for the next reporting period. First, we will continue to improve our cell-free display methodologies to enhance identification of receptor segments, particularly against recalcitrant ligands that may benefit from presentation in a format that minimizes misfolding or aggregation. Second, we will conduct proof-of-principle experiments that aim to show that high-throughput, in planta screening of signaling receptors is possible.
Impacts
What was accomplished under these goals?
Building on our work in the first year, we published our ribosome display method to identify leucine-rich repeat (LRR) segments within full-length tomato LeEIX2 and LeEIX1 that recognize Trichoderma viride ethylene-inducing xylanase (EIX) (Markou and Sarkar, Scientific Reports, 2022). We have now begun to further develop new cell-free protein engineering platform methodologies that can enable detection of interactions between receptors and recalitrant ligands. This entailsdisplaying both the receptor and the ligand in ribosome display format to avoid issues with protein expression or aggregation in cells. Finally, we have also recently begunto develop a novelin planta methodology for high-throughput screening of new receptors that enable ligand-induced signaling.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2022
Citation:
Markou, G.C., Sarkar, C.A. A cell-free approach to identify binding hotspots in plant immune receptors. Sci Rep 12, 501 (2022). https://doi.org/10.1038/s41598-021-04259-8
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Progress 05/01/20 to 04/30/21
Outputs
Target Audience:
Nothing Reported
Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?The project supported two graduate students, George Markou and Andrew Baldys, who received interdisciplinary training in biomolecular engineering and plant biology/pathology. Dr. Markou recently graduated and he has manuscript currently under review based on work he performed to map binding hotspots in plant immune receptors. Mr. Baldys is a first-year PhD student who is currently learning some of the basic molecular engineering experimental tools, like ribosome display. How have the results been disseminated to communities of interest?
Nothing Reported
What do you plan to do during the next reporting period to accomplish the goals?One of our goals in the next reporting period is to develop experimental and computational methods to further refine the resolution of the binding paratopes in immune receptors from the level of individual LRR repeats to the level of individual amino acids. We would also like develop appropriate expression systems in N. benthamiana to test the functionality (or lack thereof) of receptor mutations identified in our ribosome display experiments to demonstrate relevance in planta. We will also begin exploring plant receptor systems that have the potential to be engineered in order to alter specificity towards new targets.
Impacts
What was accomplished under these goals?
This was a challenging first year working on the project, given that it started in the middle of the pandemic and we were unable to do lab work for many months. Despite this, we were still able to develop a semi-quantitative, cell-freemethod to identify leucine-rich repeat (LRR) segments within a full-length immune receptorthat contribute to binding. This was done using ribosome display to identify LRRs within the tomato LeEIX2 and LeEIX1 that recognizeTrichoderma viride ethylene-inducing xylanase (EIX) as a case study. Leveraging the modular nature of these LRRreceptors, we applied an entropy-informed algorithm to maximize the information content in our receptor segmentation experiments to identify segments implicated in EIX binding. Unexpectedly, two distinct EIX-binding hotspots were discovered on LeEIX2 and both hotspots are shared with decoy LeEIX1, suggesting that their contrasting receptor functions are not due to differential modes of ligand binding. Given that most plant immune receptors are thought to engage targets via their LRR sequences, this approach should be of broad utility in rapidly identifying their binding hotspots.
Publications
- Type:
Journal Articles
Status:
Under Review
Year Published:
2021
Citation:
G.C. Markou and C.A. Sarkar. "A Cell-Free Approach to Identify Binding Hotspots in Plant Immune Receptors."
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