Performing Department
(N/A)
Non Technical Summary
Barley, a vital crop worldwide, suffers from a bacterial disease caused by Xt. Unfortunately, we don't fully understand how barley plants fight back against this infection at the cellular level. Plants have complex defenses, and our early findings suggest Xt doesn't infect all barley leaf cells equally. Additionally, the way the bacteria injects toxins (through a system called T3SS) may vary depending on the targeted cell.Current research on plant-bacteria battles often looks at the entire plant at once, missing the finer details happening within individual cells. This project aims to zoom in on these details, following Xt infection over time and space within barley leaves.Here's what we want to achieve:Objective 1:Understand how the fight between the bacteria and the plant's defenses unfolds in different parts of the leaf and over time.Objective 2:Compare the gene activity (the plant's "battle plan") in both the entire leaf and individual cells after infection.Objective 3:Track how the plant's defenses turn on or off in different areas of the leaf as the infection progresses.By understanding the "who, what, where, and when" of the battle within barley leaves, this project will provide valuable insights into how plants fight disease. This knowledge will be key for developing new strategies to protect barley crops from Xt and other bacterial foes.
Animal Health Component
(N/A)
Research Effort Categories
Basic
100%
Applied
(N/A)
Developmental
(N/A)
Goals / Objectives
The pathogenic bacteria,Xt, limits barley production globally, however, little information is known about how cellular responses to infection effect barely leaf colonization. During infection, plants respond to pathogens in a diverse manner. Our preliminary data demonstrates thatXtdoes not uniformly colonize inner leaf tissue and that not all cells are targeted by the bacterial Type III secretion system (T3SS). Our current understanding of plant-pathogen interactions focuses on studying plant transcriptomes at a global, whole tissue level, resulting in loss of heterogeneity that may exist during infection. This project will investigate the spatiotemporal cellular response to T3SS-targeting and the corresponding effect on bacterial colonization following these Objectives:Objective 1: To define spatiotemporal aspects of virulence and immunity on bacterial inner leaf colonization.Objective 2: To identify transcriptomic differences in plant response to Xt infection at the global and single cell level.Objective 3: Investigate spatiotemporal induction of immune or susceptible responses duringXtinfection.
Project Methods
Objective 1:To define spatiotemporal aspects of virulence and immunity on bacterial inner leaf colonization.Experiment Objective 1A: Evaluate the effect of plant innate immunity activation on Xt inner leaf colonization.The effect of basal defense response induction onXtinner leaf colonization will be evaluated. There is evidence that elicitation of innate immunity temporarily inhibits bacterial colonization[29]. To evaluate this in barley, we will imageXtcolonization on leaves treated with immunity elicitors. Two-week-old barley with dTALE-inducible GFP (cv. IGRI GFP) leaves will be submerged in 100nM flg22 solution or a mock water solution for 1 hour prior to bacterial inoculation. Barley plants will be infiltrated with mCherry taggedXtandXtDhrcTexpressing dTALE-GFP with an OD600of 0.1. Cell imaging with an Olympus laser confocal microscope FV3000 located in the Jacobs lab will be done at 0.5-, 4-, 12-, 24-, and 48-hours post inoculation (hpi). Five replicates per treatment, per bacterial strain, per time point will be imaged to better understand how immune elicitation effects spatiotemporal aspects ofXtinner leaf colonization and TALE-mediated plant cell targeting. Specifically, I will evaluate with Olympus FV3000 Cell Sense software and quantify fluorescence intensity and signal distribution of both bacterial cells and TALE-induced GFP in plant cells. For statistical analysis of bacterial spatial localization and scale, the R package spatstat will be used[30]. I will work with my collaborator and applied statistician Dr. Lopez-Nicora to apply the statistical model.Experiment Objective 1B: Evaluate the role of TALEs on Xt inner leaf colonization.There is evidence that TALEs promoteXtvirulence, but limited knowledge is available for barley[22]. To observe if manipulation of barley inner leaf tissue by TALEs contributes to spatiotemporal bacterial colonization, we will knockdown the entire gene family inXt.Recent research from our team and others applied clustered regularly interspaced short palindromic repeats interference (CRISPRi) silencing to plant pathogenic bacterial systems[31]. Using this same approach, we will design a single guide RNA (sgRNA) to target a highly conserved sequence in either the promoter or 5' UTR of TALE genes. To ensure protein expression is suppressed, we will evaluate the efficacy oftalegene silencing for each candidate sgRNA using Western blot analysis for protein abundance[31, 32].Three sgRNAs will be evaluated for their ability to silencetales inXt,and the sgRNA with the greatest knockdown will be used for the experiment.Aside from generating sgRNA, a control with theuidAgene (GUS) will be used to verify that CRISPR machinery or antibiotic resistance does not affecttalegene expression. To observe the effect thattalegene silencing has onXtinner leaf colonization, fluorescently tagged wild typeXt, XtDhrcT,XtDtale, andXt uidAcontrol will be infiltrated into barley leaves with an OD600of 0.1. Following the same time points in Objective 1A, leaf imaging will be done with confocal microscopy. Five replicates per treatment per time point will be used to evaluate the effect oftalegene silencing onXtinner leaf colonization. The same statistical modeling as Objective 1A will be applied here.Objective 2: To identify transcriptomic differences in plant response toXtinfection at the global and single cell level.Experiment Objective 2A: Identify single cell transcriptomes of barley inner leaf tissue infected with Xt.Two-week-old barley leaves (cv. IGRI GFP) will be inoculated with mCherry taggedXtdTALE-GFP,XtDhrcT, a flg22 pretreatment as above or mock solution. Bacterial samples will be infiltrated in 3cm sections at OD600= 0.1. After 24 h, protoplasts will be isolated following a protoplast isolation protocol established in the Jacobs lab. Protoplasts will be used for single cell library construction following the protocol generated by the Coaker lab[25]. The workflow for generating a single cell library will use a Chromium controller (10X Genomics) located at the Genomics Shared Resources (GSR) facilities at OSU. The Chromium controller is used for single cell partitioning to combine barcoded beads and individual protoplasts into a microfluidic chip. Protoplasts will be barcoded, and cDNA libraries will be constructed with Chromium Single Cell 3' reagent kit (10X Genomics). Library quality will be assessed with a bioanalyzer, and sequencing carried out at the Applied Microbiology Services Lab (AMSL) at OSU. Paired end 150 bp reads will be obtained with an Illumina NextSeq 2000. Data analysis will take place as a trainee visiting the Coaker lab. Data analysis will be carried out using a variety of 10X Genomics tools including Cell Ranger, Loupe Browser, and Seurat to process, count, visualize, and cluster reads.Experiment Objective 2B: Identify genes modulated in response to Xt infection using bulk RNA-seq.To complement our scRNA-seq data, we will perform a bulk dual (plant and pathogen) RNA-seq analysis on infected and mock-infected tissues using the same treatments as Objective 2A. Three replicates for each treatment will be collected 24 h after infiltration, and TRIzol will be used to extract total RNA, following manufacturers guidelines. RNA integrity will be evaluated using a bioanalyzer andsequencing will be carried out to obtain paired end 150 bp reads with an Illumina NextSeq 2000 at the OSU AMSL. Quality of reads will be assessed using FastQC (http://www.bioinformatics.babraham.ac.uk/projects/fastqc/) and low-quality reads/adapter sequences will be removed using TRIMMOMATIC v0.39. High quality reads will be aligned to Barley genome andXtgenome with STAR v2.5.0. HTSEQ will be used to count aligned reads and differential expression analysis will be performed using the DESeq2 package in R Studio.Objective 3: Investigate spatiotemporal induction of immune or susceptible responses within a barley leaf duringXtinfection.Experimental Objective 3: Develop fluorescent transcriptional reporter lines from marker genes for live-cell imaging.Three to five promoters of genes from each category of cell clusters (immune or susceptible) will be used for the generation of florescent transcriptional reporter lines. Of the genes selected, we expect some to include roles in PTI (immunity markers) or growth/development (susceptibility markers) reported from the literature and maybe novel genes. The promoters from each selected gene will be fused to a nuclear localization signal and a florescent protein (eg. GFP fusion) following the protocol outlined byZhu, Lolle [25]. Transgenic barley will be produced withAgrobacterium-mediated transformation following an established protocol in the Jacobs Lab. Independent lines will be screened for fluorescence with microscopy and the gene expression of the fluorescent protein will be evaluated with reverse-transcription quantitative PCR (RT-qPCR). Lines with adequate expression will be used for inoculation and subsequent spatiotemporal analysis.Reporter lines will be inoculated with mCherry-expressing wild-typeXt,XtDhrcT, and a mock water solution.Live cells will be imaged at the same time points used in Objective 1A&B. The number of bacterial colonies, area, and florescent intensity will be quantified with Imaris or cellSens software. Statistical analyses and spatiotemporal modeling will be done to observe the amount of marker gene expression in relation to bacterial colonization with the help of Dr. Lopez-Nicora. At various timepoints, we will quantify the proximity of marker gene expressing cells toXtcolonies. This will reveal the spatial and temporal localization of susceptible and immune response during disease progression.