Recipient Organization
UNIVERSITY OF CHICAGO
5801 SOUTH ELLIS AVE.
CHICAGO,IL 60637
Performing Department
Mol Gen/Cell Bio
Non Technical Summary
The first layer of innate immunity in plants involves perception of pathogen associated molecular patterns (PAMPs) by cell surface receptors, including FLS2, a receptor for a fragment (flg22 ) of bacterial flagellin. The purpose of this study is to determine the potential crosstalk between the PAMP response triggered by flg22 and the plant peptide phytosulfokine (PSK). There is a known interaction these two signaling pathways, but the mechanism is not clear. Specifically, I want to know the following: (1) Does PSK/PSKR affect the internalization of flg22/FLS2? (2) How are the spatial temporal dynamics of flg22/FLS2 influenced by PSK and its receptors (PSKR)? (3) Is the downstream signaling of flg22/FLS2 affected by PSK/PSKR? To answer these questions I will use a variety of mutants and microscopy techniques (TIRF & confocal) as well as radiolabeling to track the distal movement of functional labeled peptides. Biosensors will be used to monitor levels of cGMP and calcium produced in mutant plants in response to ligands. I will look at the functional interactions between flg22 and PSK pathways in Arabidopsis and in a crop plant (rice). This study offers a means of understanding how an immune signaling network is modulated by PAMPs and plant signaling peptides. With high applicability to other plant species, the system presented here could be applied to other systems and provide knowledge of different pathways that could hence be optimized for agriculture.
Animal Health Component
0%
Research Effort Categories
Basic
100%
Applied
0%
Developmental
0%
Goals / Objectives
The first layer of innate immunity in plants involves perception of pathogen associated molecular patterns (PAMPs) by cell surface receptors, including FLS2, a receptor for a fragment (flg22 ) of bacterial flagellin. The purpose of this study is to determine the potential crosstalk between the PAMP response triggered by flg22 and the plant peptide phytosulfokine (PSK). There is a known interaction these two signaling pathways, but the mechanism is not clear. Specifically, this project will address the following:(1) Deterrmine if PSK/PSKR affects the internalization of flg22/FLS2?A. Investigate the impacts of PSK/PSKR on flg22 using total internal reflection fluorescence microscopy.B. Investigate the longer-range, organ wide trafficking of both PSK and flg22 determine whether flg22 movement/trafficking is affected by PSK/PSKR, and whether PSK shows any cell to cell movement like flg22.(2) Determine if the spatial temporal dynamics of flg22/FLS2 are influenced by PSK and its receptors (PSKR).A. Determine if PSKR impacts the flg22 pathway in a manner similar to PEPR, except that it may dampen the immune response instead of amplify it since loss of PSKR enhances flg22 responses.
Project Methods
Experimental MethodsAim 1:To understand when and where PSK influences flg22 signaling, I will start by investigating potential effects in RME. I will use total internal reflection fluorescence (TIRF) and single particle trafficking to study the trafficking of the PSK and flg22 receptor-ligand complexes. I will investigate the interaction of PSK and flg22 in the Arabidopsis root tips. I will use tpst mutant lines to have more control over the PSK signaling pathway, since the only PSK present will be what I add to the system. The TIRF experiments will deploy departure assays for clathrin-mediated endocytosis from Johnson and Vert. This assay relies on dual TIRF imagining and creating a clathrin trace utilizing clathrin light chain with a GFP or RFP tag to create a reference trace for the internalization of different proteins. I will use dual channel time-lapse movies recorded sequentially. Thus, I will visually investigate one peptide at a time. I will apply both PSK and flg22 peptides: 1 with a fluorescent and without a fluorescent label, to track the internalization of each peptide. I will use the following combinations: scrambled flg22 with PSK-TAMRA, flg22 with PSK-TAMRA, nsPSK with FAM-flg22, PSK with FAM-flg22. I will analyze movies using the cmeAnalysis package in MatLab 2013b and using the custom scripts written by Johnson and Vert to determine internalization and trafficking patterns of the peptides.I expect to find a relationship between flg22 and PSK endocytic pathways, or a time dependency between the two given the fact that the two peptides trigger the creation of complexes with so many of the same membrane bound proteins and PSK signaling was inferred to affect flg22 root growth inhibition,The second goal of this objective is to investigate the longer range, organ-wide trafficking of both PSK and flg22, to determine whether flg22 movement/trafficking is affected by PSK/PSKR, and whether PSK shows any cell to cell movement like flg22. I will apply one or both fluorescently labeled PSK-TAMRA and flg22-FAM to the adaxial side of leaves of tpst plants and conduct time courses (using confocal microscopy) to determine if PSK-TAMRA/flg22-FAM are detected on the abaxial side. This will show if flg22 movement is affected by PSK and vice versa. I will use nsPSK and scrambled flg22 for nonfunctional control peptides. I will use tpst/pskr1, tpst/fls2 and tpst/pskr1/fls2 as control plants with one or more of the receptors missing and no competing endogenous PSK. I will test also for distal movement in the plant, orthostichous leaves, the stem, and roots by monitoring TAMRA and FAM fluorescence to see if there is colocalization of PSK and flg22 or if one peptide affects the pattern of movement of the other. As a complimentary method, I will apply radiolabeled peptides and use plant autoradiography to monitor changes in the pattern of the movement of flg22 in response to the addition of PSK. Peptide stability will be assessed via gel electrophoresis. Results will be analyzed using imageJ and the outputs will be reported as percent colocalization. To test the functional effect of PSK on long range trafficking of flg22, I will test if the magnitude of systemic immunity to P. syringae (assayed by pathogen growth) induced by a local flg22 treatment is affected by a prior local PSK treatment, using methods standard in our lab.Aim 2:I hypothesize that PSKR impacts the flg22 pathway in a manner similar to PEPR, except that it may dampen the immune response instead of amplify it since loss of PSKR enhances flg22 responses. First, I will investigate the potential impact that the PSKR/PSK pathway has on the cytosolic production of cGMP by monitoring the influx of calcium into the cytoplasm resulting from the flg22/FLS2 activation. After various peptide treatments, I will measure cytosol localized calcium dependent protein aequorin, which is also chemiluminescent, using the protocol from Qi et al.25 I will also investigate the cGMP production from the various CNGCs associated with PSK/PSKR and flg22/FLS2 (CNGC17 and CNGC2, respectively). I will use the FlincG27 biosensor to measure the in planta changes to cGMP in the response of tpst plants to PSK, flg22, nsPSK, and scrambled-PSK. I will replicate this experiment in rice using transiently transformed plant lines. Carbon nanofibers, which I have developed as part of my graduate work, will be used to deliver FlincG or aequorin expression plasmids. I will assay leaves and roots to determine if there is a difference between organs. I will identify potential genes whose expression pattern changes after calcium is released into the cytoplasm through CNGC channels (early and late responses of PSK/PSKR on flg22/FLS2 signaling cascade). I will use R-studio to mine (a) transcriptional profiling data obtained with tpst mutants treated with PSK and nsPSK by Dian Liu, a graduate student in the Greenberg lab, and (b) data from the literature investigating the transcriptional profiling of flg22/FLS2 signaling in both Arabidopsis and rice plants. Dian is investigating the early and late responses to PSK without an induced immune response. Additionally, there are published RNA-seq results of Arabidopsis exposed to flg22. Highly expressed genes from early and late responses induced by PSK and flg22, respectively that I will confirm with qRT-PCR. Lastly, I will test their expression upon treatment with PSK and flg22, separate and together, and in pskr1 and fls2 plants to test which genes are affected by the presence of one or more ligands.Production of the Carbon NanofibersCarbon based nanofibers with a lateral spacing (pitch) of 10 or 35 μm were made in the Center for Nanophase Material Science at Oak Ridge National Lab using nickel catalyst dots across the top of a silicon wafer via electron beam lithography, metal evaporation, and conventional lift-off using the method described by (Melechko et al., 2009). For the lithography step, the silicon wafers were coated with PMMA resist and exposed using a JEOL 9300FS electron beam lithography system and developed in a solution of xylene:isopropyl alcohol. Electron beam lithography is necessary to define the catalyst dots with diameters between 200 nm and 500 nm. After developing the resist and cleaning with a 6-second exposure to oxygen plasma, electron beam evaporation was used to deposit a thin nickel film on the patterned resist. Sequential sonication in acetone and isopropyl alcohol is used to remove the underlying resist layer, leaving catalyst dots in the desired pattern behind. Nanofiber growth was then conducted using a plasma-enhanced chemical vapor deposition chamber (PEVCD) using an acetylene/ammonia mixture. PECVD growth parameters were optimized to control the length and taper of the nanofibers. After fabricating the VACNFs, their geometry was assessed using scanning electron microscopy.EffortsThe results from this work will be presented in classroom and outreach activities conducted by the PD Jessica Morgan and the mentor Dr. Jean Greenberg.EvaluationTo work towards the goals of the project, I will confer with my primary and collaborative mentors bi-weekly to discuss my current progress. I will attend conferences annually to give progress updates and receive feedback from the community, write progress reports for theUSDA and meet with my thesis committee to evaluate my progress and provide feedback.TimelineY1: Analyze RNA-seq data from Dian Liu and literature. Complete TIRF experiments and singleparticle tracking analysis for Aim 1, write and submit paper. Gather materials for project.Y2: Complete experiments for Aim 1. Perform experiments in aim 2, specifically investigate calciumand cGMP production and other studies with Arabidopsis and rice, write and submit paper.Y3: Finish experiments/writing that may be interrupted by COVID-19.