Grant Information
| Knowledge Area | Subject of Investigation | Field of Science | Percent |
|---|---|---|---|
| 212 - Pathogens and Nematodes Affecting Plants | 999 - Citrus, general/other | 1080 - Genetics (excludes breeding) | 100% |
The disease Huanglongbing (HLB), caused by Candidatus Liberibacter asiaticus (CLas) is devastating the U.S. citrus industry. Effective sources of genetic resistance have not been identified and horticultural control practices can be costly and ineffective. To address the need for enhanced resistance in citrus germplasm, efforts in this proposal combine expertise in gene editing technologies, plant tissue culture and phloem responsive gene identification to develop a gene editing pipeline with capabilities to modify the expression of multiply phloem and defense associated genes. We anticipate that altered expression or activity of phloem defense responses that are not well activated during CLas infection will enhance resistance levels. Proposed studies will apply highly-efficient CRISPR-based editing tools to a protoplast regeneration system that will subsequently feed into a statistically robust germplasm screening system for the assessment of HLB resistance. Additional studies will mine current and ongoing genomic and transcriptional findings to identify CLas responsive defense promoters and motifs that can be targeted for transcriptional reprogramming via these editing tools. Combined, the proposed gene-editing systems will provide a pipeline to move gene discovery findings into edited citrus germplasm that can then be readily screened for CLas resistance, with identified resistance move to the field testing phase.
The disease Huanglongbing (HLB), caused by Candidatus Liberibacter asiaticus (CLas) is devastating the U.S. citrus industry. Effective sources of genetic resistance have not been identified and horticultural control practices can be costly and ineffective. To address the need for enhanced resistance in citrus germplasm the goals of this proposal focus on the development of gene editing technologies, plant tissue culture and phloem responsive gene identification to develop a gene editing pipeline with capabilities to modify the expression of multiply phloem and defense associated genes. We anticipate that altered expression or activity of phloem defense responses that are not well activated during CLas infection will enhance resistance levels. Proposed studies will apply highly-efficient CRISPR-based editing tools to a protoplast regeneration system that will subsequently feed into a statistically robust germplasm screening system for the assessment of HLB resistance. Additional studies will mine current and ongoing genomic and transcriptional findings to identify CLas responsive defense promoters and motifs that can be targeted for transcriptional reprogramming via these editing tools. Combined, the proposed gene-editing systems will provide a pipeline to move gene discovery findings into edited citrus germplasm that can then be readily screened for CLas resistance, with identified resistance move to the field testing phase. Specific objectives include:
Two multiplexed CRISPR systems will be set up and evaluated in citrus for manipulating target gene expression. The first uses a highly efficient multiplexed Cas12a system for promoter editing. This system uses the ribonucleoprotein delivery of the Cas12a protein and guide RNAs directly into protoplasts, providing a highly efficient, transient and transgene-free editing system. The second system will use a multiplexed CRISPR system to simultaneously alter the expression of multiple target genes using a robust dCas9 based activation/repression system, CRISPR-Act3.0. Both systems will be transfected into citrus protoplasts derived from embryogenic Hamlin and Valencia calli. Additional studies will utilize gene discovery and bioinformatic approaches to identify host defense genes and their corresponding promoter domains that are differentially expressed in response to Candidatus Liberibacter asiaticus (CLas). These genes will represent the targets for transcriptional reprograming using the described gene editing systems. The efficiency of target gene/promoter modification will be assessed by sequencing and transcriptional analyses. Calli derived from transfected protoplasts will subsequently be used for the regeneration of citrus embryos and seedlings. Subsequent promoter edited defense associated genes will be assessed for promoter activity via in silico sequence analysis and in situ promoter activation studies in both tissue culture and regenerated citrus germplasm.
Target Audience
Results from this project were targeted to the scientific community through presentations at the annual ECDRE directors meeting as well as national and international plant science meetings. Additional outreach was through the submission and publication of journal publications.
Changes / Problems
Nothing Reported
Training & Professional Development
Training opportunities in citrus tissue culture, gene promoter characterization, gene editing development and gene analysis were provided for for four undergraduate students and three postdoctoral students.
Dissemination Streams
Results from these studies have been published or are being prepared for publication in peer-reviewed journals. Corresponding gene sets have been deposited in the NCBI BioProject. Project findings have also been presented at several meetings including the International Institute of Tropical Agriculture (IITA), CGIAR,Nairobi, Kenya;the Plant Genome Stability and Change-EMBO Workshop,Olomouc, Czech Republic;the Plant Center Spring Symposium, University of Georgia; and the Innovative Genomics Institute, UC-Berkeley.
Next Reporting Steps
Nothing Reported
Target Audience
Results from this project have been accomplished through journal publications as listed under products.
Changes / Problems
COVID restrictions, including hiring and space restrictions significantly delayed the onboarding of key personnel for this project thus slowing our progress. In addition, the recalcitrant nature of citrus to tissue culture regeneration required us to develop and apply new CRISPR editing and activation systems to promote and improve plant regeneration systems needed for this project. Because of these delays we are requesting a 2nd no cost extension of 12 months. With the new systems for enhanced citrus regeneration and the needed personnel in place we anticipate completing the project within the requested extension.
Training & Professional Development
Training opportunities in citrus tissue culture, gene editing development and gene analysis were provided for one graduate student and two postdoctoral students.
Dissemination Streams
Results have been disseminated primarily through the publications listed.
Next Reporting Steps
As outlined, using a new CRISPR-Combo system we have achieved a significant advancement in efforts to enhance citrus regeneration. The requested 2nd NCE will allow us to further apply this system to complete efforts to modify the phloem expression of citrus genes associated with HLB resistance and/or susceptibility. Our initial CRISPR-Combo morphogenic gene activation studies have focused on Carrizo citrange due to its amenability to regeneration. With the additional requested 2nd NCE, efforts will examine more recalcitrant yet economically important citrus varieties including sweet orange varieties Hamlin and Valencia. Specific studies will investigate the use of morphogenic gene activation to enhance plant regeneration in edited protoplasts, embryogenic callus and hypocotyl sections from designated sweet orange varieties as outlined in Objective 1. Efforts will also investigate the application of the CRISPR-Combo system to introduce gene modifications that suppress HLB as proposed in Objectives 2 and 3. Additional, efforts will explore the previously established embryogenic protoplast and calli systems for morphogenic gene activation and editing using the developed CRISPR-Combo ribonucleoprotein system (RNP). RNP deliver will occur via PEG or liposomes to protoplasts and through biolistic delivery to the shoot apical meristems and hypocotyl cell layers of targeted sweet orange varieties. We anticipate that outcomes from the requested 2nd NCE will result in the development of powerful CRISPR-Cas systems for enhanced citrus regeneration and phloem targeted gene-editing. The application of this system is critical to achieving the proposed genome modifications to citrus germplasm in an efficient and routine manner for subsequent testing against CLas infection. <br><br>
<br>What was accomplished under these goals? Objective 1. Development of multiplexed gene editing and gene activation methods in citrus. Task 1. Establish citrus protoplast transformation systems. A stable callus maintenance, protoplast and transformation system was established as reported in previous progress reports. Task 2, Develop multiplexed Cas12a system for promoter editing. CRISPR-CAS12a ribonucleoprotein (RNP) mediated mutagenesis has been established as reported previously. In addition, we developed an RNP system for CRISPR-Combo, a new gene editing system that can both direct targeted mutations and activate specific gene transcripts. CRISPR-Combo was used to investigate the activation of citrus morphogenic genes to enhance the regeneration of gene edited cells, which is a major obstacle in citrus transformation. Twenty different CRISPR-Combo systems targeting ten different morphogenic genes and two CLas associated genes, CsNPR3 and CsLOB1, were created and tested for their impact on citrus regeneration and target gene editing. Results from this study revealed significant enhancements to citrus culture regeneration and are outlined below in Objective 3, task 2. Future efforts will explore the previously established embryogenic protoplast and calli systems for morphogenic gene activation and editing using non-transgene CRISPR-Combo RNPs. RNP deliver will occur via PEG or liposomes delivery to protoplasts and through biolistic delivery to the shoot apical meristems and hypocotyl thin cell layers of targeted sweet orange varieties. Task 3, Develop CRISPR-Act3,0 for tissue-specific activation of target genes in phloem. CRISPR-Act3.0 enhancer constructs targeting phloem gene specific promoters identified in Objective 2 are currently being tested within an Arabidopsis model system for phloem activation. Objective 2. Identification and validation of phloem and defense promoter specific sequence motifs for use in transcriptome reprogramming. Task 1. In silico identification of target gene promoter sequences. As previously reported, in silico and meta-analysis of previous transcriptome studies on HLB sensitive, tolerant and resistant citrus cultivars were performed, identifying gene transcripts that are significantly altered in response to HLB. This information will be used in future CRISPR-Combo constructs that will target the transcriptional activation of both morphogenic genes for enhanced plant regeneration as well as the identified HLB response genes. Task 2. Expression analysis and validation of identified target genes. As stated above we are applying the CRISPR-Combo system as a means to enhance citrus regeneration from culture. The enhanced regeneration observed with this system has the potential to overcome the recalcitrance of citrus to regenerate and allow for the screening of edited target genes as proposed. Objective 3. Validation of gene editing systems for the targeted transcriptional reprogramming of host defense responses. Task 1. qPCR analysis of promoter edited protoplasts and cam for regenerated germplasm. Gene editing directed by a CRISPR-Cas12a ribonucleoprotein (RNP) system as well as a transgene directed CRISPR-Combo system have been established within citrus protoplasts and hypocotyl stem sections. In addition, an RNP system for the CRISPR Act3.0 activation system was also validated in vitro for assembly and gene editing activation. Task 2 Regeneration and in situ expression analysis of promoter edited earn and germplasm. The major goal of this project is to develop gene-editing systems that promote the modified expression of phloem expressed target genes associated with defense against and or susceptibility to Clas. While we have made significant progress on developing multiplexed gene editing systems for this study and identifying phloem specific genes for targeted gene editing a significant obstacle has been the inability to regenerate citrus from transformed protoplasts or callus tissues as many citrus cultivars are recalcitrant to regeneration efforts. To address this bottleneck, we incorporated a newly developed CRISPR system, termed CRISPR-Combo, that can direct both targeted gene editing and the activation of morphogenic genes that enhance plant regeneration. Using this approach twenty different CRISPR-Combo systems targeting ten different morphogenic genes and two Clas associated genes, CsNPR3-defense and CsLOB1-susceptibility, were created and tested for their impact on citrus regeneration and target gene editing. For these studies Carrizo citrange (Carrizo citrange trifoliate hybrid, Citrus sinensis "Washington" sweet orange X Poncirus trifoliata) was selected for CRISPR-Combo testing using Agrobacterium-mediated stable transformation. The regeneration rate of Citrus explants was assessed and compared among various CRISPR-Combo constructs that targeted different morphogenic genes. Significantly higher regeneration rates, characterized by the emergence of dense clustered shoots were observed upon activation of morphogenic genes CsSERK1L, CsWUS3, and CsBBM3. PCR amplification and Sanger sequencing were subsequently used to confirm the gRNA directed disruption of targeted Clas associated genes. These findings demonstrate the potential of the CRISPR-Combo system to overcome citrus associated regeneration bottlenecks. One drawback of the enhanced shoot proliferation associated with the CRISPR-Combo activation of target morphogenic genes was that the majority of the regenerated shoots were abnormal, did not produce roots and were not suitable for micrografting onto rootstock material. To address this issue modifications were made to the culture media, ultimately removing any growth hormones that are normally included to promote plant regeneration. On media lacking growth hormone normal shoot development was observed on hypocotyl sections transformed with CRISPR-Combo targeting CsSERK1L morphogenic gene activation while controls fail to develop any shoots. These results demonstrate that the activation of targeted morphogenic genes in the absence of additional growth hormones is sufficient to produce normal shoot production suitable for rooting and/or micrografting. Combined these results represent a substantial improvement in the ability to apply gene editing tools to improve citrus germplasm. <br><br><b>Publications</b><br>
Target Audience
Results from this project have been presented at the annual ECDRE directors meeting. Additional outreach has been through the submission and publication of journal outlines as listed under products.
Changes / Problems
COVID19 restrictions, including hiring and space restrictionsslowed some experiments anddelayed the hiring of some project personnel. Due to these issues we have requested a no cost extension of one year.
Training & Professional Development
Training opportunities in citrus tissue culture and gene analysis were providied for two postdoctoral students.
Dissemination Streams
Nothing Reported
Next Reporting Steps
We have established citrus protoplasts and CRISPR-RNP gene editing systems. Additional studies will continue developing CRISPR tools for tissue (phloem) and pathogen response specific gene editing. In addition, as noted above one main bottleneck has been thetime and efficiency associated with regenerating transformed citrus cells. Through on going and planned experiments we are adapting the CRISP-Combo system to citrus for both targeted gene editing and enahnced plant regeneration. This will involve the screening of ~20 putative morphogenic genes for assemsement of their impact on citrus regeneration. <br><br>
<br>What was accomplished under these goals? Objective 1. Development of multiplexed gene editing and gene activation methods in citrus. Task 1. Establish citrus protoplast transformation systems.A stable callus maintenance procedure including both solid plate culture and liquid suspension culture has been developed. Citrus protoplast isolation and polyethylene glycol (PEG) mediated protoplast transfection has been established. Citrus cells from embryogenic callus cultures were used to produce protoplasts via standard isolation methods. Reporter constructs carrying the marker genes were subsequently used to test PEG mediated protoplast transfection. The average transfection efficiency was determined to be 30-90% depending on the construct used. Parameters such as protoplast numbers, target DNA concentration and incubation time of the transfection were subsequently optimized in order to promote greater transformation efficiency. Task 2. Develop multiplexed Cas12a system for promoter editing.?CRISPR-CAS12a ribonucleoprotein (RNP) mediated mutagenesishas been established. A CRISPR-Cas12a based RNP delivery system has been tested on the protoplasts derived from citrus callus cultures. The wild type Cas12a enzyme (LbCas12a) and the crRNA scaffolds based on the CRISPR-LbCpf1 system were designed to target specific citrus genes. These components were pre-assembled with a molar ratio of 1:2 to form the RNP complex.TheRNP complex was delivered to citrus protoplast through PEG mediated protoplast transfection developed in Task 1 above.Afterthe transfection, protoplasts were incubated in the dark for 48 hr followed by genomic DNA isolation and restriction fragment length polymorphism (RFLP) assay. Different concentrations (0.1, 0.01 and 0.001 μM) of RNP complex were tested in the targeted mutagenesis of citrus protoplast. The result shows that the target site has been successfully mutated by the CRISPR-Cas12a based RNP delivery system with a 0.1 μM concentration of RNP displaying a nearly 90% editing efficiency, which indicates the CRISPR-Cas12a based RNP delivery system is a powerful tool for citrus genome editing. In addition, we have developed an RNP system for CRISPR-Combo, a new gene editing system that can both direct targeted mutations and activate specific gene transcripts. In particular we are interested in activating specific citrus morphogenic genes to improve the rate of plant regeneration. CRISPR-Combo assembly requires polypeptides for the Cas nuclease, SunTag amplification scaffold and transcriptional activation (TAD) as well as gRNAs targeting gene activation and editing. For these studies, we established a recombinant CRISPR-Combo protein expression system for the Cas nuclease, SunTag, and TAD polypeptides using the pET27b (+) vector and His-tag purification system per the manufacturer's protocol. Associated gRNAs are designed and synthesized for insertion into T7 transcription cassettes. These gRNA cassettes can be used for in vitro T7 RNA transcription and purification using commercial kits. Purified Cas, SunTag, TAD and gRNAs are mixed in an optimal ratio of 1:6:100:2 and incubated for 1 hour in 1X NEBuffer 3.1 at room temperature prior to testing. Initial in vitro testing has used a plasmid vector harboring a gRNA targeted restriction enzyme sequence with associated PAM site. Results have demonstrated the assembly and activity of this new RNP system. Ongoing studies are aimed at optimizing this system and moving it into citrus cells to examine its impact on plant regeneration, see Objective 3, task 2 below. Task 3. Develop CRISPR-Act3.0 for tissue-specific activation in phloem.CRISPR-Act3.0 enhancer constructs targeting citrus phloem gene specific promoters identified in Objective 2 are currently being developed.This system uses a defective dCas9 enzyme to target transcriptional activator domains to gRNA specified promoter sequences. Furthermore, additional phloem specific promoters have beenmoved into the CRISPR-Act3.0 T-DNA vectors so as to drive dCas9 expression in a phloem specific manner. Objective 2. Identification and validation of phloem and defense promoter specific sequence motifs for use in transcriptome reprogramming. Task 1.In silicoidentification of target gene promoter sequences. Meta-analysis of previous transcriptome studies on HLB sensitive, tolerant and resistant citrus cultivars were performed to identify gene transcripts that are significantly altered in response to HLB. This analysis has identified a set of defense associated genes that are significantly upregulated in resistant and tolerant cultivars but not in sensitive cultivars. This information is being used to generate phloem expressed CRISPR-Act3.0 constructs targeted at increasing the expression levels of select candidate genes in the phloem of HLB sensitive cultivars. Task 2. Expression analysis and validation of identified target genes.As stated above in Objective 1, Task 3, identified phloem specific promoters are being investigated as a means to drive the expression of the CRISPR Act3.0 dCas9 enzyme as a means to enhance target gene expression specifically in phloem tissues. To assess this approach more rapidly we developed a series of Arabidopsis transgenic lines with marker genes driven by phloem promoters of varying strength. Current studies are using these marker genes to both assess the ability of CRISPR-Act3.0 to enhance gene expression in a phloem specific manner as well as examine the optimal promoter regions for targeting the Act3.0 gRNAs. Objective 3. Validation of gene editing systems for the targeted transcriptional reprogramming of host defense responses. Task 1. qPCR analysis of promoter edited protoplasts and calli for regenerated germplasm.As described above a protoplast transformation / gene editing process using the CRISPR-Cas12a ribonucleoprotein system was shown to be nearly 90% effective in editing a target gene sequence in citrus protoplasts. Additional studies have identified unique defense and phloem specific gene promoter sequences for use in subsequent multiplex CRISPR Act3.0 activation. Efforts in this task are ongoing and are focused on further investigating the efficacy of the Cas12a RNP system as well as establishing the Act3.0 gene activation system in our protoplast system. Task 2. Regeneration andin situexpression analysis of promoter edited calli and germplasm.Reliably embryogenesis and embryo germination protocols for citrus embryogenic lines have been successfully established. Globular embryos were derived from embryogenic callus after three to four weeks on solid MT-O media at 25°C in light. Once globular embryos formed, individual embryo could be transferred onto embryo germination media for development to the heart-shaped embryo stage and finally development to plantlets. To promote the formation of roots, plantlets were then transferred onto root induction media or micro-grafted onto Carrizo citrange rootstock. We also note a key bottleneck in this project is the time and efficiency associated with regenerating transformed plants. To address this concern, we are currently investigating the use of a newly developed CRISPR system, termed CRISPR-Combo, thatcan direct both targeted gene editing and the activation of morphogenic genes that enhance plant regeneration.In rice, CRISPR-Combo transgene directed morphogenic gene activation was shown to result in the regeneration of seedlings in hormone-free culture medium, with 83% of the regenerated plants carrying the targeted gene edit. No rice plants were regenerated in non-CRISPR-Combo controls. We are currently using aCarrizo citrange (Carrizo citrange trifoliate hybrid,Citrus sinensis"Washington" sweet orange XPoncirus trifoliate) transformation system to test the impact of activating a set of known plant morphogenic genes on citrus regeneration with the goal of using this information to enhance the efficiency and speed of our protoplast and embryogenic callus transformations. <br><br><b>Publications</b><br>
Target Audience
Nothing Reported
Changes / Problems
COVID19 restrictions, including hiring and space limitations slowed some experiments and has delayed bringing ona third post-doctoral student to work with Dr. Dardick, USDA-ARS, on gene and promoter analysis as well as asist with the production of gene editied citrus lines. Applicants for this position are currently being screened and the University returned to 100% occupancy this past June. These changes should rectify the notedlimitations.
Training & Professional Development
Training opportunities in citrus tissue culture and gene analysis were providied for two postdoctoral students.
Dissemination Streams
Nothing Reported
Next Reporting Steps
Efforts will continue as outlined under the objectives and timeline of the proposal. Specific ongoing and future efforts will focus on expanding protoplast gene editing efforts and demonstrating the regeneration of edited protoplasts into seedlings. Additional efforts will perform in planta characterizations of identified phloem and HLB induced promoters and promoter motifs as well as the use of the Act3.0 editing system to promote the expression of defense associated genes found to be induced in response to HLB. <br><br>
<br>What was accomplished under these goals? Objective 1. Development of multiplexed gene editing and gene activation methods in citrus.? Task 1. Establish citrus protoplast transformation systems. A stable callus maintenance procedure including both solid plate culture and liquid suspension culture has been developed.Citrus embryogenic callus lines (Citrus sinensis) under investigation in this study include: Hamlin 89 (H89), Hamlin 19 (H19), Valencia 17 (V17) and Pineapple 17 (Pa17). To make these cell lines more capable for protoplast isolation, citrus cell suspension culture were developed by crushing calli of each line in 50 mL liquid MT basal medium and incubating at 25°C in dark with 120 rpm shaking and a 21-day subculture cycle. Among these four cell lines, Hamlin cells (H89 and H19) developed much better than V17 and Pa17 lines. It is observed that, after 3-month's culture, Hamlin cells scatter evenly in suspension media while cells of Valencia and Pineapple lines stayed in clumps. Moreover, Hamlin lines, H89 line especially, display a high growth rate (doubling time about 4 to 5 days according to a rough estimationbased on fresh weight). These features make H89 a desired citrus cell line for protoplast isolation and further protoplast-based experiments. Citrus protoplast isolation and polyethylene glycol (PEG) mediated protoplast transfection has been established.Citrus cells from line H89 were used to produce protoplasts via standard isolation methods that include Cellulase R10 and Macerozyme R10 treatments that were capable of producing ~5×106live protoplasts per 0.8g of callus. A reporter construct carrying the GFP marker gene was subsequently used to test PEG mediated protoplast transfection. The average transfection efficiency was determined to be ~26%. Parameters such as protoplast numbers, target DNA concentration and incubation time of the transfection were optimized in order to promote greater transformation efficiency. Task 2. Develop multiplexed Cas12a system for promoter editing.? CRISPR-Cas12a ribonucleoprotein (RNP) mediatedtargeted mutagenesishas been established. A CRISPR-Cas12a based RNP delivery system has been tested on the protoplasts derived from citrus cells of line H89. The wild type Cas12a enzyme (LbCas12a, IDT Inc. USA) and the crRNA scaffolds based on the CRISPR-LbCpf system and targeting the citrus gene (CsPH5, GenBank ID 102616096) were pre-assembled with a molar ratio of 1:2 to form the RNP complex. The targeted gene edit is designed to disrupt a unique restriction enzyme site as a marker for identification.TheRNP complex was delivered to citrus protoplast through PEG mediated protoplast transfection which is the sameasthe transfection of reporter GFP construct described above.Afterthe transfection, protoplasts were incubated in the dark for 48 hr followed by restriction fragment length polymorphism (RFLP) assay. Different concentrations (0.1, 0.01 and 0.001 μM) of RNP complex were tested in the targeted mutagenesis of citrus protoplast. The result shows that the target site has been successfully mutated by the CRISPR-Cas12a based RNP delivery system and a 0.1 μM of RNP complex in the delivery system performs nearly 90% editing efficiency, which indicates the CRISPR-Cas12a based RNP delivery system is a powerful tool for citrus genome editing. Task 3. Develop CRISPR-Act3.0 for tissue-specific activation in phloem. CRISPR-Act3.0 enhancer constructs targeting the promoters of defense associated genes identified in objective 2 are currently being developed. Objective 2. Identification and validation of phloem and defense promoter specific sequence motifs for use in transcriptome reprogramming.? Task 1.In silicoidentification of target gene promoter sequences. Target defense genes and promoter motifs identified for gene editing.Meta-analysis of previous transcriptome studies on HLB sensitive, tolerant and resistant citrus cultivars were screened to identify gene transcripts that are significantly altered in response to HLB. This analysis has identified a set of defense associated genes that are significantly upregulated in resistant and tolerant cultivars but not in sensitive cultivars. We are currently working to increase the expression levels of these candidate genes in the phloem of HLB sensitive cultivars using the CRISPR Act 3.0 activation system. In addition, to identify potential promoter motifs needed to drive defense gene expression within the plants vascular phloem tissue, the site of HLB infection,we have first identified phloem specific genes via a meta-analysis of previous studies. Similar analysis were also performed on non-phloem specific genes, as a negative control, to confirm the specificity of phloem identified motifs. We have identified two motifs that show conservation within the promoters of putative phloem expressed citrus genes. These motifs will be examined for their ability to confer phloem expression in citrus. Task 2. Expression analysis and validation of identified target genes.? The above identified promoter motifs for both defense and phloem expression are currently being moved into expression vectors to test their ability to selectively drive the transcription of marker genes in citrus. In addition, these motifs will also be stacked to create a synthetic promoter to drive transcription of marker and defense genes in phloem, particularly in response to pathogen invasion. Objective 3. Validation of gene editing systems for the targeted transcriptional reprogramming of host defense responses. Task 1. qPCR analysis of promoter edited protoplasts and calli for regenerated germplasm. As described above a protoplast transformation / gene editing process using the CRISPR-Cas12a ribonucleoprotein system was shown to be nearly 90% effective in editing a target gene sequence in citrus protoplasts.Additional studies have identified unique defense and phloem specific gene promoter sequences for multiplex subsequent CRISPR Act3.0 activation. Efforts in this task are ongoing and will focus on further investigating the efficacy of the Cas12a RNP system as well as establishing the Act3.0 gene activation system in our protoplast system. Task 2. Regeneration andin situexpression analysis of promoter edited calli and germplasm. Reliably embryogenesis and embryo germination protocols for citrus embryogenic lines have been successfully established.Globular embryos were derived from embryogenic callus after three to four weeks' incubation on solid MT-O media (MT basal media without any growth regulator) at 25°C in light. Once globular embryos formed, individual embryo could be transferred onto embryo germination media for development to the heart-shaped embryo stage and finally development to plantlets. To promote the formation of roots, plantlets were then transferred onto root induction media. After 2 to 3 weeks of root induction, plantlets with well-exhibited roots were able to be transferred to additional tissue culture media and then to soil. <br><br><b>Publications</b><br>