Non Technical Summary
Huanglongbing disease ("HLB") caused by the bacterium Candidatus Liberibacter asiaticus ("CLas") has killed millions of citrus trees and reduced average yield in Florida by 50%, costing billions in lost revenue.CRISPR is the most powerful tool for rapid breeding of new HLB-resistant citrus trees, but has to be preceded by identifying specific gene-editing targets. The identification of target genes has been challenging because of poor understanding of the HLB pathogenesis mechanism and technical limitations of HLB-citrus, protein-interaction screening methods.A powerful new tool to address these limitations is TurboID, an engineered biotin ligase that can label its proximal proteins when it is expressed in cells. The proposed project addressed this opportunity by employing TurboID-mediated proximity labeling to identify citrus proteins that interact with effector proteins that are critical for HLB CLas infection.During Phase I research, we produced a high-confidence list of CLas Sec-dependent effector ("SDE")-interacting citrus proteins by using TurboID coupled with mass spectrometry. In Phase II, we will use the discovered proteins as gene-editing targets for CRISPR precision breeding, to develop citrus varieties that disrupt the effector-citrus interactions, leading to genetic resistance to HLB.To that end, we will perform three objectives. The first is CRISPR-editing of promising SDE-interacting genes and generating non-transgenic CRISPR-edited citrus varieties. The second is testing CRISPR-edited plants for resistance to HLB through graft inoculation and proteomics analysis. The third is to commence field trials.We anticipate this project will develop non-GMO, HLB-resistant citrus trees that will be sold to citrus growers.
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Goals / Objectives
GoalThe goal of this Project isto cure HLB disease by creating resistant trees using CRISPR precision breeding technology. The identification of the target genes has been challenging due to poor understanding of the HLB-citrus pathogenesis mechanism and technical limitations of HLB-citrus protein-interaction screening methods. Thus during Phase I, weidentifyiedHLB susceptibility/resistance genes by discovering disease-promoting bacterial effector-interacting proteins using TurboID-mediated proximity labeling approach. In Phase II, we willcreatenon-GMO citrus plants resistant to HLB using CRISPR precision breeding, focusing on multiplexed CRISPR systems.This research presents the opportunity to test the use of the new TurboID tool to quickly and accurately identify potential disease susceptibility/resistance genes in vivo, and to test the benefits of multiplexed CRISPR gene editing to quickly create new beneficial traits for HLB-resistant crop varieties. This research also presents an opportunity to solve an immediate agricultural problem, HLB, which has caused devastating economic loss to the citrus industry in the US.Technical ObjectivesObjective 1. CRISPR-editing of promising SDE-interacting genes and generation of non-transgenic CRISPR-edited citrus varietiesWe will create SDE-interacting gene constructs focused on the three priority genes found during the Phase I research. We will then perform polyethylene glycol (PEG)-mediated transfection of isolated sweet orange protoplast using the SDE-interacting gene constructs. The sweet orange protoplast will then be regenerated and screened for the desired edit.This proposed approach is technically feasible because of the citrus transfection of Soilcea, and the expertise of collaborator Dr. Zhiyong Wang who pioneered TurboID analysis in plants. Given Dr. Wang's expertise, we have strong confidence that the targeted SDE-interacting genes are key genes in HLB disease progression. The principal investigator, Dr. Quinton Allen, has a strong background in citrus molecular biology and citrus transformation. The Soilcea lab is also the first lab to successfully transfect and regenerate Valencia sweet orange protoplast. Thus, the proposed project using CRISPR to edit promising SDE-interacting genes is feasible.Objective 2. Screening of CRISPR-edited citrus plants for resistance to HLB.The regenerated CRISPR-edited plants will then go through extensive testing for HLB resistance. The Soilcea lab has experience in three disease inoculation methods: reverse grafting, tradition grafting, and psyllid inoculation. Reverse grafting involves grafting CRISPR-edited or wild type control young leaves onto HLB-positive trees. Traditional grafting involves grafting HLB-positive budwood onto CRISPR-edited or wild type trees. Psyllid inoculation involves placing HLB-carrying phyllids in an enclosed space with CRISPR-edited and wild type trees. Given this experience, we do not anticipate any challenges with this objective.Objective 3. Field trial of HLB-resistant and canker-resistant CRISPR-edited trees.During this objective, Soilcea will take CRISPR-edited trees into the field. Soilcea recently purchased a farm close to its lab, and has an approved permit request from the USDA to commence field trials. Soilcea will thus be prepared for field trials toward the end of this Phase II project.
Objective 1. CRISPR-editing of promising SDE-interacting genes and generation of non-transgenic CRISPR-edited citrus varietiesWe will use a tRNA-based single guide and multiplex CRISPR system to edit the 2 high priority and 5 total genes individually and simultaneously through multiplexing. The Phase I project yielded a high value list of probable HLB-resistant genes. This project will initially focus on the highest priority genes discussed above, but there are other secondary priority genes that may be explored in this project.1-1. Selection of target mutations for multiplex CRISPR editingAchieving a high level of HLB resistance will likely require mutations in multiple gene targets. For our final product, we will integrate the best performing gene edits into our citrus varieties. We anticipate that multiplexing certain combinations of gene targets for knocking out may cause adverse phenotypes in the plants or even cause lethality. Thus, finding the ideal combination of gene targets to multiplex will require extensive investigation and testing. We will prioritize the single target loci that show high disease resistance from our traditional grafting inoculation screening. We will also select groups of genes to multiplex that function in the same pathway or biological process, where on their own may not produce a resistant phenotype due to complementation, but in combination may induce a resistance.1-2. Plasmid construction of multiplex CRISPR constructsFor multiplex CRISPR gene editing, we have generated a new tRNA-based multiplex CRISPR plasmid vector, pSMulti-Non-Transgenic (pSMNT), modified from the published multiplex CRISPR system (Huang et al., 2020), which will be used to synthesize the multiplexed sgRNA sequences and ligate them into the pSMNT vector through Golden Gate cloning.1-3. Protoplast transfection and regeneration of non-transgenic CRISPR-edited citrus varietiesFor the non-transgenic gene editing, Soilcea has designed a non-transgenic multiplex-compatible Cas9-sgRNA vector system (pSMNT). This approach ensures that plants will not be transgenic because this approach does not utilize Agrobacterium tumefaciens, and there are no DNA-based mechanisms present to allow insertion into the genome. Soilcea has confirmed no foreign DNA present in protoplast regenerants in previous experiments. For transfection, protoplasts will be isolated from suspension cultures and deliver the Cas9-multiplexed sgRNA plasmid constructs into protoplast cells (Guo et al., 2005; Omar & Grosser, 2007; Huang et al., 2020).The next step in the proposal will be to regenerate transfected cells. The transfected cells will be regenerated using combinations of liquid and solid media with varying balances of auxin and cytokinin hormones (Omar & Grosser, 2007).All citrus plants that regenerate will be assayed for successful editing.Objective 2. Screening of CRISPR-edited citrus plants for resistance to HLB.The nature of the arms race between plants and pathogens is highly complex. Pathogens can use different pathogenesis pathways depending on the surrounding environment and are able to evolve new mechanisms to invade resistant hosts. Thus, eliminating a single pathogenesis pathway may not provide long-term durable disease-resistance if the pathogen evolves a new pathogenesis route. We will continually screen CRISPR-edited citrus plants generated during Phase II to confirm our preliminary results and identify additional HLB-resistant target loci that are involved in different pathways. For the HLB-resistant plant screening, Soilcea will conduct 1) traditional grafting inoculations to confirm reduced CLas bacterial titer level and to evaluate disease symptom development and 2) expose the trees to populations of CLas carrying psyllids to recreate a natural infection under high disease pressure to determine their level of resistance with a more natural infection.2-1. Traditional Grafting for HLB resistance evaluationGraft inoculations will be the first screen for HLB-resistance of our promising single and multiplexed edited citrus varieties. The screening process will follow the Plant Improvement Germplasm Evaluation Guidelines approved by researchers from Arizona, California, Florida and Texas (CRDF, 2018). For the grafts, 6 to 12 month-old candidate plants will be inoculated with CLas by grafting 2 buds from an HLB-infected Valencia tree (Hilf & Lewis, 2016).2-2. Proteomic and transcriptomic analysis of CRISPR-edited treesAt the end of symptom evaluation, Soilcea will perform RNA-seq and quantitative proteomic analyses of the wild-type controls and edited plants, with and without CLas inoculation. The stem and leaf tissues will be harvested and stored at -80°C. Aliquots of tissues will be used for RNA extraction, library preparation, and RNA-sequencing, which will be carried out by a next-generation sequencing service such as Novogene Inc. For proteomic analysis, the total protein will be extracted and digested with trypsin using the S-Trap sample preparation kit (PROTIFI inc). The tryptic peptides will be labeled with the tandem mass tag (TMT, Thermo Fisher Scientific) 18-plex reagents. The labeled peptides will be mixed together, cleaned up, and analyzed on the Orbitrap Eclipse mass spectrometer. The 18-plex TMT reagents will allow us to mix and quantitatively compare 18 biological samples. These RNA-seq and proteomic analyses will illustrate the transcriptional and posttranscriptional pathways mediated by different effector targets. The results may identify additional genes involved in HLB susceptibility, and guide our double or triple mutant combinations.Objective 3. Field trial of HLB-resistant CRISPR-edited trees.For each promising new variety, Soilcea will begin clonal propagation for field trials. The initial field trial will be conducted at Soilcea's farm that is located within 11 miles of its lab. Citrus trees used in the field trial will be initially exposed to psyllids to initiate the CLas infection prior to the field trial to shorten the timeline of symptom development and elevated titer levels in susceptible trees. Since psyllid carrying CLas is pervasive in Florida, infection will continue to naturally occur after the start of the trial. During the trial, a minimum of 8 gene-edited trees for each promising variety and 8 wild-type controls will be planted in a randomized complete block design.The following tree measurements will be taken at the beginning and end of the trial and every 6 months in between: trunk diameter, canopy diameter/volume, and tree height. Foliar disease ratings, including photographic documentation, will be conducted each fall, spring, and summer. Percentage of leaves with symptoms will be scored on a 1-5 scale. General canopy thickness and color will also be rated 1-5. At the same time as foliar disease ratings, leaves and fibrous roots will be randomly collected for qPCR detection of CLas. Additionally, nutrient concentrations will be measured in the summer in 4-6 month old spring flush leaves. Finally, if the citrus trees are producing fruit , fruit yield, size, weight, and quality will also be recorded. Soilcea expects initial field trial data before the end of Phase II research, but expects to continue and expand these field trials into Phase IIb research.Through these field trials, Soilcea will statistically determine whether gene-edited varieties are HLB or canker resistant, whether this HLB and canker resistance translates to improved overall tree health and growth, and ultimately whether citrus growers get a return on investment from HLB and canker resistance trees with increased yield.