Performing Department
(N/A)
Non Technical Summary
Rice is a staple for ~half of the world's population and is an important contributor to the U.S. economy. Bacterial leaf streak (BLS), caused by the bacteriumXanthomonas oryzaepv. oryzicola (Xoc), is a threat to rice production in most rice-growing regions of the world. Xoc has not yet been found in the U.S.and isa federal select agent or considered to be a threat to rice health in the U.S. Xoc, like most other bacteria in the same genus, carry proteins called transcription activator-like effectors (TALEs) that they inject into rice cells. TALEs can travel to the rice nucleus and activate rice genes beneficial for the bacteria. However, rice is not defenseless against TALE-carrying Xoc. A U.S. rice variety, Carolina Gold Select, carries an immune receptor called Xo1 that detects TALEs injected into rice cells and launches a defense response. If Xo1 is present in rice, it can stop the pathogen from causing BLS. Even more importantly, Xo1 can detect many types of TALEs, making it a promising source of broad-spectrum resistance against many types of Xoc strains, but also other Xanthomonas pathogens of crop plants. Interestingly, there are some TALEs from different species of Xanthomonas that evade detection by Xo1. Furthermore, a subset of Xoc strains have evolved a new type of shortened TALE (truncTALE) that, when injected into the rice cell, completely shuts down Xo1's ability to detect TALEs and defend against Xoc. Researchers don't yet know how Xo1 detects some but not all TALEs to trigger immunity, or how truncTALEs inhibit Xo1 to shut down immunity and facilitate disease. This project aims to uncover what is occurring between these proteins. Findings from this project will support the broader, future goal of engineering Xo1 to detect an even wider variety of TALEs from other agriculturally relevant Xanthomonas pathogens or to resist defense suppression by truncTALEs.To investigate how Xo1 detects TALEs and truncTALEs inhibit Xo1, we would like to know what components of the TALEs and truncTALEs are required for their respective activities. I will first perturb predicted regions of importance or swap regions between TALEs and truncTALEs. Xoc carrying each of these mutant proteins will be inoculated onto Carolina Gold Select rice to measure the level of disease or resistance. I will take a similar approach of perturbing predicted important regions of Xo1 and seeing whether those mutations impact Xo1's ability to detect TALEs or be inhibited by truncTALEs. In plants, I will also identify how Xo1 interacts with TALEs and truncTALEs. By identifying parts of TALE, truncTALE, and Xo1 proteins that are important for disease or immunity, and better understanding how each component might interact with each other, we can shed light on how Xo1 operates. Not only will these findings illuminate an uncharacterized immune receptor mechanism, but it will also allow us to conceptualize how we might engineer Xo1 for enhanced disease resistance against Xoc and other Xanthomonas pathogens.
Animal Health Component
0%
Research Effort Categories
Basic
100%
Applied
(N/A)
Developmental
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Goals / Objectives
Preface: Xanthomonas oryzae pv. oryzicola (Xoc) causes bacterial leaf streak of rice. Xo1 is a rice immune receptor that confers resistance against Xoc. TALEs are effectors of Xoc that trigger Xo1 immunity. TruncTALEs are truncated variants of TALEs that inhibit Xo1 immunity.Goal [1]: to identify how Xo1 detects or is inhibited by pathogen effectors.- Objective [1.1]: Identify TALE and truncTALE regions required foractivation and inhibition of Xo1-mediated immunity.- Objective [1.2]: Determine whether TALEs interact with Xo1 and truncTALEs disrupt this interaction.- Objective [1.3]: Determine whether nuclear localization of TALEs and truncTALEs is required for Xo1 induction and inhibition.- Objective [1.4]: Determine whether TALE DNA-binding is required for Xo1 immunity.- Objective [1.5]: Determine importance of Xo1 regions in immunity.- Objective [1.6]: Determine importance of Xo1 nuclear localization on immunity.- Objective [1.7]: Collect disease specimens and isolate Xoc strains. Pathotype collected strains.Goal [2]: to support mentored research, collaboration, and professional development experiences to position the PD for a career in plant pathology.- Objective [2.1]: mentor REU students and undergraduate research assistants- Objective [2.2]: work with collaborators on structural characterization of the rice immune receptor and/or the pathogen effectors via cryo-EM- Objective [2.3]: present at conferences, publish manuscripts, and maintain collaborations
Project Methods
Methods:[1] test ability ofTALEs and theirvariants to trigger or inhibit Xo1- synthesize TALE variants with standard cloning techniques (Gibson assembly, Golden Gate, Gateway)and transform into pathogen- confirm TALE expression via Western blotting- inoculate Xoc carrying TALE variants on rice lines carrying Xo1 and test virulence- measure leaf lesion lengths caused by Xoc strains and analyze differencesvia ANOVA and Tukey HSDtest[2] test potential Xo1-TALE, Xo1-truncTALE, Xo1-TALE-truncTALEinteractions and determine Xo1 regions important for triggering immunity viatransgenicrice lines- standard rice transgenic development protocol- producelines carrying Xo1tagged with TurboID for proximity labeling approach- test function of transgene by inoculation with Xoc and leaf lesion measurements as above- identify any captured interactors with TurboID pipeline and LC/MS-MS-produce lines with tagged Xo1 deletion/swap mutants to identify regions important for immunity-test function of transgene by inoculation with Xoc and leaf lesion measurements as above[3]test Xo1-TALE,Xo1-truncTALE, and Xo1-TALE-truncTALEinteractions and determine Xo1 regions important for triggering immunity viatransient assay- transfect rice protoplasts with plasmids encoding TALE, truncTALE,and/or Xo1via standard rice protoplast protocol- detect/measure fluorescence forlocalization experiments in protoplasts- measure cell death for immune output assays in protoplasts[4] test DNA binding of the variant TALEs- EMSA to determine whether TALEs bind target DNA (established previously by lab)- quantify apparent dissociation constant betweenTALE variants and DNA via EMSAEvaluation:- mentorship milestones: mentor summer REU student, mentor honors thesis undergraduate student- professional development milestones: present in at least one conference per year, maintain new collaborations- communication milestones: publish at least two papers from research generated in project, present at student seminars and lab meetings