Source: SOIL CULTURE SOLUTIONS, LLC submitted to
IDENTIFICATION OF CANDIDATUS LIBERIBACTER ASIATICUS EFFECTOR INTERACTOME BY PROXIMITY LABELING COUPLED WITH PROTEOMICS IN CITRUS
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
COMPLETE
Funding Source
SMALL BUSINESS GRANT
Reporting Frequency
Annual
Accession No.
1025822
Grant No.
2021-33530-34507
Cumulative Award Amt.
$100,000.00
Proposal No.
2021-01527
Multistate No.
(N/A)
Project Start Date
Jul 1, 2021
Project End Date
Feb 28, 2023
Grant Year
2021
Program Code
[8.2]- Plant Production and Protection-Biology
Recipient Organization
SOIL CULTURE SOLUTIONS, LLC
3802 SPECTRUM BLVD STE 142H
TAMPA,FL 336129223
Performing Department
(N/A)
Non Technical Summary
Huanglongbing disease ("HLB") has killed millions of citrus trees and reduced average yield in Florida by 33%, costing the citrus industry 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 addresses this opportunity by employing TurboID-mediated proximity labeling to identify citrus proteins that interact with effector proteins that are critical for HLB bacterial infection. We will then 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 two objectives. The first objective is generating effector-TurboID expressing transgenic citrus plants. We will use HLB bacterium Sec-dependent effector ("SDE") proteins proven to cause HLB-disease symptoms, including SDE1 and SDE15.The second objective is identifying SDE-interacting citrus proteins by TurboID coupled with mass spectrometry. We will produce a high-confidence list of SDE-interacting citrus proteins through data analysis.We anticipate this project will identify gene-editing targets that we will then use to develop non-GMO, HLB-resistant citrus trees that will be sold through citrus nurseries to citrus growers.
Animal Health Component
100%
Research Effort Categories
Basic
(N/A)
Applied
100%
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
20109991040100%
Knowledge Area
201 - Plant Genome, Genetics, and Genetic Mechanisms;

Subject Of Investigation
0999 - Citrus, general/other;

Field Of Science
1040 - Molecular biology;
Goals / Objectives
We aim to cure HLB disease by creating resistant trees using CRISPR precision breeding technology, but must first identify specific gene editing targets. The identification of the target genes has been challenging because of poor understanding of the HLB-citrus pathogenesis mechanism and technical limitations of HLB-citrus protein-interaction screening methods.The technical goal is identifying HLB susceptibility/resistance genes by discovering disease-promoting bacterial effector-interacting proteins using TurboID-mediated proximity labeling approach (Phase I) and then creating non-GMO citrus plants resistant to HLB using CRISPR precision breeding, focusing on multiplexing CRISPR systems (Phase II).Objective 1. Generation of SDE-TurboID expressing transgenic citrus.We will create SDE-TurboID constructs focused on SDE1 and SDE15 because of their proven involvement in HLB disease but poor understanding of the pathogenesis mechanism. We will then perform Agrobacterium-mediated transformations of Carrizo epicotyl using the SDE-TurboID constructs. Transgenic Carrizo shoots will then be regenerated and selected from the epicotyls to examine the SDE-citrus protein interactions by TurboID.Objective 2. Identification of CLas SDE-interacting proteins by TurboID- mediated proximity labeling coupled with MS analysis.TurboID-MS experiments consist of 5 steps. Biotin treatment -> Affinity purification of biotinylated proteins -> Peptide digestions -> MS analysis -> Quantitative analysis of MS spectrums.
Project Methods
Objective 1. Generation of SDE-TurboID expressing transgenic citrus.1) Plasmid construction.The Soilcea research team will perform the SDE-TurboID plasmid construction. To investigate the CLas SDE-citrus interactions, Soilcea has chosen SDE1 and SDE15 because of their proven involvement in HLB disease. Soilcea will use two different types of promoters (Overexpression and Inducible; Table 1). For overexpression, we will use the gateway compatible pTurboID-101 plasmid which contains a 35S promoter-driven TurboID-YFP-HA tag at the C-terminal of the protein of interest (Kim et al., 2019). The pTurboID-101 vector has been developed by Dr. Zhiyong Wang's lab and proven to work with plant species. For inducible TurboID expression, we will use an estradiol-inducible pMDC7 vector. These two vector systems will be provided to Soilcea by Dr. Zhiyong Wang.The SDEs contain signal peptides at N-terminal regions in their immature forms. Thus, the SDE sequences without signal peptides and the stop codons will be cloned into the pENTR/SD/D-TOPO entry vector and subcloned into the pTurboID-101 vector. For inducible promoter constructs, SDE1-TurboID and SDE15-TurboID fragment sequences from SDE1-TurboID and SDE15-TurboID plasmids, respectively, will be PCR amplified and cloned into the pENTR/SD/D-TOPO entry vector. Successful entry constructs will be subcloned into pMDC7 vectors to generate estradiol-inducible SDE-TurboID constructs. YFP protein sequences will be used as a negative control.The final 6 plasmid constructs will be transformed into Agrobacterium strain EHA105.2) Transformation of TurboID in citrus and generation of transgenic plants.Soilcea will use Carrizo rootstock epicotyl for Agrobacterium-mediated transformations. Soilcea will perform all the transformation and generation of transgenic plants. We will follow a well established method for Agrobacterium-mediated citrus transformation with some modifications (Orbovi? and Grosser, 2015). Successful transgenic shoots from Carrizo epicotyl explants will be screened using antibiotics-resistance, YFP fluorescence, and immunoblot assay. The selected transgenic shoots will be transferred to the growth medium for further growth. Juvenile transgenic shoots that reach a height of 20 mm will be collected for TurboID-mediated biotin labeling experiments. In addition to the shoots for the labeling experiments, at least 5 individual transgenic trees for each construct will be maintained for future research in Phase II (for testing TurboID in adult trees and tissue-specific TurboID experiments).* Objective 2. Identification of CLas SDE-interacting proteins by TurboID-mediated proximity labeling coupled with MS analysis.1) TurboID-mediated proximity labeling-MS analysis of SDE proteins.Soilcea will perform the biotin treatment, Streptavidin-affinity purification of biotinylated proteins, and peptide digestions by the methods published by Kim et al. (2019). For biotin treatment, we will pool at least 5 individual transgenic Carrizo shoots (about 2 mm in height) per repeat experiment. The shoots will be infiltrated with 50 μM biotin solution using needleless syringe and incubated for 3 hours in the solution. This biotin concentration and the incubation time were adapted from previously optimized conditions (Kim et al., 2019). For inducible TurboID constructs, we will induce the SDE-TurboID expression by the 5 μM estradiol treatment 1 day prior to the biotin treatment. To facilitate the research progress, a subset of the biotin-treated samples will be sent to Dr. Zhiyong Wang's lab for processing.After biotin treatment, total proteins will be extracted using the extraction buffer and non-labeled free biotin in the protein extracts will be removed by desalting columns. The cleared protein extracts will be incubated with Streptavidin-conjugated beads for affinity purification of biotin-labeled proteins. The affinity-purified biotinylated proteins will be digested into peptides by Trypsin and subsequently purified by C18 column chromatography. The purified peptides will be sent to Dr. Zhiyong Wang's lab and be analyzed on a Q-Exactive HF hybrid Quadrupole-Orbitrap mass spectrometer (Thermo Fisher) equipped with an Easy LC 1200 UPLC liquid chromatography system (Thermo Fisher) that are located in the mass spectrometry facility of the Carnegie Institution for Science at Stanford University.At least 4 biological replicates and about 60 MS samples will be analyzed by Dr. Zhiyong Wang's lab.2) Quantitative data analysis.To identify the SDE-interacting proteins, Dr. Zhiyong Wang's lab will perform LFQ analysis of the SDE-TurboID samples. The MSMS files will be searched against the C. clementina (v1.0) and C. sinensis (v1.1) databases from Phytozome (https://phytozome.jgi.doe.gov/) using MaxQuant software. We will use a 1% FDR cutoff at the protein, peptide, and modification levels to minimize false positives. The first peptide precursor mass tolerance will be set at 20 ppm, and MS/MS match tolerance will be set at 20 ppm. Search criteria will include a static carbamidomethylation of cysteines and variable modifications of oxidation on methionine residues, acetylation at the N-terminus of proteins, and biotinylation (+226.078 Da) on N-terminus of proteins or lysine residues. The match between runs function will be enabled with a match time window of 0.7 min. The minimum peptide length will be seven amino acids, and a minimum Andromeda score will be set at 40 for modified peptides. Peptide intensities from 4 biological replicates of control and SDE-TurboID samples will be obtained. The protein intensities will be calculated by averaging the intensity of each peptide detected from the protein sequence. The proteins identified in at least 3 replicates in the SDE-TurboID samples and have biotin modification will be selected for further quantitative analysis. Average of the protein intensity from replicates will be compared with the control samples. Downstream statistical analysis and graphic presentation will be performed using the Perseus software (version 1.6.2.1) (Tyanova et al., 2016) and Microsoft Excel.The quantification results from the biological repeats will be categorized by 1) stringent interactors (proteins that are detected at least three times exclusively in the SDE-TurboID samples), 2) moderately stringent interactors (proteins that show higher levels in the SDE-TurboID samples than the control or that are detected less than three times in SDE-TurboID samples), and 3) non-interactors (proteins that are detected but not enriched in the SDE-TurboID samples). Each SDE-interactome dataset will be compared with data from the different promoter types. Furthermore, the interacting proteins detected from both promoter datasets will be considered high confident interactors.3) Protein function and interacting network prediction.Dr. Zhiyong Wang's lab will provide the lists of putative SDE-interacting proteins to Soilcea after MS analysis. The resulting SDE interactorswill be compared with orthologs to predict their functions and involvement in disease response. Soilcea together with Dr. Zhiyong Wang's lab will find Arabidopsis orthologs of the SDE-interacting proteins by BLAST search. The list of Arabidopsis orthologs will be used to build a functional protein association network using the STRING program (https://string-db.org/) because the Arabidopsis protein network has been extensively investigated. The categorization of interactors and functional prediction will provide a clear view of target networks of SDE1 and SDE15 and will produce a manageable list of SDE-interacting proteins for the Phase II research to conduct CRISPR experiments to develop citrus varieties with resistance to HLB.

Progress 07/01/21 to 02/28/23

Outputs
Target Audience: If this project is successful, many stakeholders will benefit. The technical benefit will be identifying HLB susceptibility/resistance genes and creating citrus varieties resistant to HLB. The economic benefit will be the revitalization of the citrus industry with HLB resistant trees that prevent early tree death, increase yields to pre-HLB levels, provide cost savings to citrus growers who no longer need to spend so much on inputs, and promote environmental benefits from reduced pesticide use. The Federal Government and other researchers will benefit with a proof of concept of using TurboID to identify protein interactions in vivo and using CRISPR to create disease-resistant crops. Citrus growers are Soilcea's direct customer. Soilcea's disease-resistant tree will benefit citrus growers affected by HLB by increasing yield and reducing costs. The average yield loss due to HLB is 33% or 100 boxes per acre ($873 per acre at 145 trees and $8.72 per box), and $783 per acre is spent each year to control HLB through preventative measures, such as pesticides, antibiotics, and fertilizers. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?During this project, we hired and trained a new lab technician. A guest lecture was also provided at the University of South Florida. How have the results been disseminated to communities of interest?Soilcea has an advisory board of citrus industry experts. Ron Edwards (Chairman) is currently the President and CEO of Evans Properties, Inc., which operated 25,000 acres of citrus fields before the spread of HLB, previously was the COO of Tropicana Product, and was a founding investor and managing member of the South Beach Beverage Corporation (SOBE Brand), which was sold to PepsiCo, and a founder and managing member of Blue Buffalo Pet Products, which was acquired by General Mills Corporation. The other advisory members include Dr. Harold Browning, President of Premier Citrus Apz, former Chief Operating Officer of the Citrus Research and Development Foundation, and former Center Director and Professor at the Citrus Research and Education Center, University of Florida. Fran Becker is the current Senior Vice President of Peace River Citrus Products. Clay Pederson is Managing Director of Agromillora Florida. Dave Crumbly is the Vice President, Agricultural Services at Florida's Natural Growers. Soilcea has also attended the International Congress of Citrus Nurseries in Visalia, CA, touring California nursery facilities, such as Tree Source and Wonderful. In the last year, we've presented our research in front of the Citrus Research and Development Foundation, the Florida's Naturals Board, the Florida Citrus Mutual, and the California Citrus Research Board. Soilcea has also entered a propagation agreement with Agromillora, a leading citrus nursery. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? During the USDA SBIR Phase I project Soilcea identified citrus proteins that interact with HLB causing bacterial effector proteins by employing TurboID-mediated proximity labeling. To that end, both objectives were achieved, and all milestones were accomplished. The first objective was generating effector-TurboID expressing transgenic citrus plants. Bacterial Sec-dependent effector ("SDE") proteins proven to cause HLB-disease symptoms, including SDE1 and SDE15, were used for this experiment. To generate gene constructs that express the SDE1 and SDE15 proteins fused with the TurboID biotin ligase, the Entry cloning system was used along with 3 different plant expression vector systems (pTurboID-101, pMDC7, and pMDC32). Soilcea generated 12 different plasmid constructs and transformed those constructs into Agrobacterium EHA105 strain for citrus transformation. Between 2,000 to 7,500 citrus Carrizo stem segments with each construct were transformed. YFP positive shoots regenerated from the transformed stem segments 2-3 months after the transformation. The second objective was identifying SDE-interacting citrus proteins by TurboID coupled with mass spectrometry. Using shoots obtained from the first objective, all 9 TurboID expressing transgenic shoots were confirmed to be producing the fusion protein in immunoblotting assays. As the TurboID-mediated proximity labeling system has not been tested in citrus, we optimized the biotin concentration for proximity labeling and the estradiol treatment time for pMDC7-based transgenic shoots. We found that 50 uM biotin treatment produced maximum labeling efficiency and that overnight estradiol treatment produced high expression of TurboID fusion proteins without affecting the tissue viability in pMDC7-based transgenic shoots. With these optimized conditions, we prepared estradiol/biotin treated tissue samples that were sent to the Carnegie Institution for Science and sample preparation for MS is in progress. The Carnegie Institution then provided MS analysis, and further research into the function of the genes generated a list of 2 priority putative SDE1- and SDE15-interacting citrus target proteins as well as a list of 3 additional lower priority target proteins. These effector-interacting proteins provide critical information about HLB pathogenic mechanisms and the genes encoding these proteins will be CRISPR gene editing targets for HLB-resistant citrus variety development in this Phase II project.

Publications


    Progress 07/01/21 to 06/30/22

    Outputs
    Target Audience:The target audience and users of this technology will be the citrus industry. During this reporting period, Soilcea has attended two citrus conferences to engage in direct outreach with citrus growers and other citrus industry participants. Soilcea has also presented at a meeting organizedby the Citrus Research Development Foundation in front of citrus growers, politicians, and other stakeholders. Soilcea also presented in front of the California Citrus Research Board. Soilcea has also created an advisory board made up of citrus industry participants and held its first advisory board meeting. These activities were done to explain thetechnical benefits ofidentifying HLB susceptibility/resistance genes and creating citrus varieties resistant to HLB. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?During this project, our scientists trained an intern from University of South Florida and a Research Associate. For professional development, our scientists attended citrus industry conferences. The Tampa Bay Technology Incubator, which we are a part of, also provides seminars on various business and science related topics. How have the results been disseminated to communities of interest?As discussed in the target audience section, we have engaged in significant outreach to the citrus community, including attending two citrus industry conferences, presenting to a meeting of citrus industry participants, and organizing and presenting to our own advisory board. What do you plan to do during the next reporting period to accomplish the goals?In the next reporting period, we plan to use the target genes discovered in Phase I research to begin developing new CRISPR-edited, HLB-resistant citrus varieties. We will also continue to engage the citrus community and plan to train another intern.

    Impacts
    What was accomplished under these goals? We accomplished both objectives. The first objective wasgenerating effector-TurboID expressing transgenic citrus plants. We used bacterial Sec-dependent effector ("SDE") proteins proven to cause HLB-disease symptoms, including SDE1 and SDE15. To generate plasmid constructs that express the SDE1 and SDE15 proteins fused with TurboID, we used the Entry cloning system and 3 different plant expression vector systems (pTurboID-101, pMDC7, and pMDC32). We generated 9 different plasmid constructs and transformed those constructs into Agrobacterium EHA105 strain for citrus transformation. We have transformed between 2,000 to 7,500 citrus Carrizo stem segments with each construct. We have produced YFP positive shoots regenerated from the transformed stem segments 2~3 months after the transformation. The second objective wasidentifying SDE-interacting citrus proteins by TurboID coupled with mass spectrometry. Using shoots obtained from the first objective, we confirmed that all 9 TurboID expressing transgenic shoots produced the fusion proteins in immunoblotting assays. As the TurboID-mediated proximity labeling system has not been tested in citrus, we optimized the biotin concentration for proximity labeling and the estradiol treatment time for pMDC7-based transgenic shoots. We found that 50 uM biotin treatment produced maximum labeling efficiency and that overnight estradiol treatment produced high expression of TurboID fusion proteins without affecting the tissue viability in pMDC7-based transgenic shoots. With these optimized conditions, we prepared estradiol/biotin treated tissue samples. These samples have been sent to the Carnegie Institution for Science for MS analysis. The Carnegie Institution performed MS analysis and identified 9 promising target genes which will be used as target for CRISPR editing to create HLB-resistant citrus varieties.

    Publications